Inbred corn line PHE67

ABSTRACT

A novel inbred maize line designated PHE67 and seed, plants and plant parts thereof. Methods for producing a maize plant that comprise crossing inbred maize line PHE67 with another maize plant. Methods for producing a maize plant containing in its genetic material one or more traits introgressed into PHE67 through backcross conversion and/or transformation, and to the maize seed, plant and plant part produced thereby. Hybrid maize seed, plant or plant part produced by crossing the inbred line PHE67 or a trait conversion of PHE67 with another maize line. Inbred maize lines derived from inbred maize line PHE67, methods for producing other inbred maize lines derived from inbred maize line PHE67 and the inbred maize lines and their parts derived by the use of those methods.

FIELD OF THE INVENTION

This invention relates generally to the field of maize breeding,specifically relating to an inbred maize line designated PHE67.

BACKGROUND OF THE INVENTION

The goal of plant breeding is to combine, in a single variety or hybrid,various desirable traits. For field crops, these traits may includeresistance to diseases and insects, resistance to heat and drought,reducing the time to crop maturity, greater yield, and better agronomicquality. With mechanical harvesting of many crops, uniformity of plantcharacteristics such as germination, stand establishment, growth rate,maturity, plant height and ear height, is important. Traditional plantbreeding is an important tool in developing new and improved commercialcrops.

SUMMARY OF THE INVENTION

According to the invention, there is provided a novel inbred maize line,designated PHE67 and processes for making PHE67. This invention relatesto seed of inbred maize line PHE67, to the plants of inbred maize linePHE67, to plant parts of inbred maize line PHE67, and to processes formaking a maize plant that comprise crossing inbred maize line PHE67 withanother maize plant. This invention also relates to processes for makinga maize plant containing in its genetic material one or more traitsintrogressed into PHE67 through backcross conversion and/ortransformation, and to the maize seed, plant and plant part produced bysuch introgression. This invention further relates to a hybrid maizeseed, plant or plant part produced by crossing the inbred line PHE67 oran introgressed trait conversion of PHE67 with another maize line. Thisinvention also relates to inbred maize lines derived from inbred maizeline PHE67 to processes for making other inbred maize lines derived frominbred maize line PHE67 and to the inbred maize lines and their partsderived by the use of those processes.

DEFINITIONS

Certain definitions used in the specification are provided below. Alsoin the examples that follow, a number of terms are used herein. In orderto provide a clear and consistent understanding of the specification andclaims, including the scope to be given such terms, the followingdefinitions are provided. NOTE: ABS is in absolute terms and % MN ispercent of the mean for the experiments in which the inbred or hybridwas grown. PCT designates that the trait is calculated as a percentage.% NOT designates the percentage of plants that did not exhibit a trait.For example, STKLDG % NOT is the percentage of plants in a plot thatwere not stalk lodged. These designators will follow the descriptors todenote how the values are to be interpreted. Below are the descriptorsused in the data tables included herein.

ABTSTK=ARTIFICIAL BRITTLE STALK. A count of the number of “snapped”plants per plot following machine snapping. A snapped plant has itsstalk completely snapped at a node between the base of the plant and thenode above the ear. Expressed as percent of plants that did not snap.

ALLELE. Any of one or more alternative forms of a genetic sequence.Typically, in a diploid cell or organism, the two alleles of a givensequence typically occupy corresponding loci on a pair of homologouschromosomes.

ALTER. The utilization of up-regulation, down-regulation, or genesilencing.

ANTHESIS. The time of a flower's opening.

ANT ROT=ANTHRACNOSE STALK ROT (Colletotrichum graminicola). A 1 to 9visual rating indicating the resistance to Anthracnose Stalk Rot. Ahigher core indicates a higher resistance. Data are collected only whensufficient selection pressure exists in the experiment measured.

BACKCROSSING. Process in which a breeder crosses a hybrid progeny lineback to one of the parental genotypes one or more times.

BARPLT=BARREN PLANTS. The percent of plants per plot that were notbarren (lack ears).

BORBMN=ARTIFICIAL BRITTLE STALK MEAN. The mean percent of plants not“snapped” in a plot following artificial selection pressure. A snappedplant has its stalk completely snapped at a node between the base of theplant and the node above the ear. Expressed as percent of plants thatdid not snap. A high number is good and indicates tolerance to brittlesnapping.

BRENGMN=BRITTLE STALK ENERGY MEAN. The mean amount of energy per unitarea needed to artificially brittle snap a corn stalk. A high number isgood and indicates tolerance to brittle snapping.

BREEDING. The genetic manipulation of living organisms.

BREEDING CROSS. A cross to introduce new genetic material into a plantfor the development of a new variety. For example, one could cross plantA with plant B, wherein plant B would be genetically different fromplant A. After the breeding cross, the resulting F1 plants could then beselfed or sibbed for one, two, three or more times (F1, F2, F3, etc.)until a new inbred variety is developed. For clarification, such newinbred varieties would be within a pedigree distance of one breedingcross of plants A and B. The process described above would be referredto as one breeding cycle.

BRTSTK=BRITTLE STALKS. This is a measure of the stalk breakage near thetime of pollination, and is an indication of whether a hybrid or inbredwould snap or break near the time of flowering under severe winds. Dataare presented as percentage of plants that did not snap. Data arecollected only when sufficient selection pressure exists in theexperiment measured.

CELL. Cell as used herein includes a plant cell, whether isolated, intissue culture or incorporated in a plant or plant part.

CLDTST=COLD TEST. The percent of plants that germinate under cold testconditions.

CLN=CORN LETHAL NECROSIS. Synergistic interaction of maize chloroticmottle virus (MCMV) in combination with either maize dwarf mosaic virus(MDMV-A or MDMV-B) or wheat streak mosaic virus (WSMV). A 1 to 9 visualrating indicating the resistance to Corn Lethal Necrosis. A higher scoreindicates a higher resistance. Data are collected only when sufficientselection pressure exists in the experiment measured.

COMRST=COMMON RUST (Puccinia sorghi). A 1 to 9 visual rating indicatingthe resistance to Common Rust. A higher score indicates a higherresistance. Data are collected only when sufficient selection pressureexists in the experiment measured.

CROSS POLLINATION. A plant is cross pollinated if the pollen comes froma flower on a different plant from a different family or line. Crosspollination excludes sib and self pollination.

CROSS. As used herein, the term “cross” or “crossing” can refer to asimple X by Y cross, or the process of backcrossing, depending on thecontext.

D/D=DRYDOWN. This represents the relative rate at which a hybrid willreach acceptable harvest moisture compared to other hybrids on a 1 to 9rating scale. A high score indicates a hybrid that dries relatively fastwhile a low score indicates a hybrid that dries slowly.

DIPERS=DIPLODIA EAR MOLD SCORES (Diplodia maydis and Diplodiamacrospora). A 1 to 9 visual rating indicating the resistance toDiplodia Ear Mold. A higher score indicates a higher resistance. Dataare collected only when sufficient selection pressure exists in theexperiment measured.

DIPLOID PLANT PART. Refers to a plant part or cell that has the samediploid genotype as PHE67.

DIPROT=DIPLODIA STALK ROT SCORE. Score of stalk rot severity due toDiplodia (Diplodia maydis). Expressed as a 1 to 9 score with 9 beinghighly resistant. Data are collected only when sufficient selectionpressure exists in the experiment measured.

DRPEAR=DROPPED EARS. A measure of the number of dropped ears per plotand represents the percentage of plants that did not drop ears prior toharvest. Data are collected only when sufficient selection pressureexists in the experiment measured.

D/T=DROUGHT TOLERANCE. This represents a 1 to 9 rating for droughttolerance, and is based on data obtained under stress conditions. A highscore indicates good drought tolerance and a low score indicates poordrought tolerance. Data are collected only when sufficient selectionpressure exists in the experiment measured.

EARHT=EAR HEIGHT. The ear height is a measure from the ground to thehighest placed developed ear node attachment and is measured incentimeters.

EARMLD=GENERAL EAR MOLD. Visual rating (1 to 9 score) where a 1 is verysusceptible and a 9 is very resistant. This is based on overall ratingfor ear mold of mature ears without determining the specific moldorganism, and may not be predictive for a specific ear mold. Data arecollected only when sufficient selection pressure exists in theexperiment measured.

EARSZ=EAR SIZE. A 1 to 9 visual rating of ear size. The higher therating the larger the ear size.

EBTSTK=EARLY BRITTLE STALK. A count of the number of “snapped” plantsper plot following severe winds when the corn plant is experiencing veryrapid vegetative growth in the V5-V8 stage. Expressed as percent ofplants that did not snap. Data are collected only when sufficientselection pressure exists in the experiment measured.

ECB1LF=EUROPEAN CORN BORER FIRST GENERATION LEAF FEEDING (Ostrinianubilalis). A 1 to 9 visual rating indicating the resistance topreflowering leaf feeding by first generation European Corn Borer. Ahigher score indicates a higher resistance. Data are collected only whensufficient selection pressure exists in the experiment measured.

ECB2IT=EUROPEAN CORN BORER SECOND GENERATION INCHES OF TUNNELING(Ostrinia nubilalis). Average inches of tunneling per plant in thestalk. Data are collected only when sufficient selection pressure existsin the experiment measured.

ECB2SC=EUROPEAN CORN BORER SECOND GENERATION (Ostrinia nubilalis). A 1to 9 visual rating indicating post flowering degree of stalk breakageand other evidence of feeding by second generation European Corn Borer.A higher score indicates a higher resistance. Data are collected onlywhen sufficient selection pressure exists in the experiment measured.

ECBDPE=EUROPEAN CORN BORER DROPPED EARS (Ostrinia nubilalis). Droppedears due to European Corn Borer. Percentage of plants that did not dropears under second generation European Corn Borer infestation. Data arecollected only when sufficient selection pressure exists in theexperiment measured.

ECBLSI=EUROPEAN CORN BORER LATE SEASON INTACT (Ostrinia nubilalis). A 1to 9 visual rating indicating late season intactness of the corn plantgiven damage (stalk breakage above and below the top ear) causedprimarily by 2^(nd) and/or 3^(rd) generation ECB larval feeding beforeharvest. A higher score is good and indicates more intact plants. Dataare collected only when sufficient selection pressure exists in theexperiment measured.

EGRWTH=EARLY GROWTH. This is a measure of the relative height and sizeof a corn seedling at the 2-4 leaf stage of growth. This is a visualrating (1 to 9), with 1 being weak or slow growth, 5 being averagegrowth and 9 being strong growth. Taller plants, wider leaves, moregreen mass and darker color constitute higher score. Data are collectedonly when sufficient selection pressure exists in the experimentmeasured.

ELITE INBRED. An inbred that contributed desirable qualities when usedto produce commercial hybrids. An elite inbred may also be used infurther breeding for the purpose of developing further improvedvarieties.

ERTLDG=EARLY ROOT LODGING. The percentage of plants that do not rootlodge prior to or around anthesis; plants that lean from the verticalaxis at an approximately 30 degree angle or greater would be counted asroot lodged. Data are collected only when sufficient selection pressureexists in the experiment measured.

ERTLPN=EARLY ROOT LODGING. An estimate of the percentage of plants thatdo not root lodge prior to or around anthesis; plants that lean from thevertical axis at an approximately 30 degree angle or greater would beconsidered as root lodged. Data are collected only when sufficientselection pressure exists in the experiment measured.

ERTLSC=EARLY ROOT LODGING SCORE. Score for severity of plants that leanfrom a vertical axis at an approximate 30 degree angle or greater whichtypically results from strong winds prior to or around floweringrecorded within 2 weeks of a wind event. Expressed as a 1 to 9 scorewith 9 being no lodging. Data are collected only when sufficientselection pressure exists in the experiment measured.

ESTCNT=EARLY STAND COUNT. This is a measure of the stand establishmentin the spring and represents the number of plants that emerge on perplot basis for the inbred or hybrid.

EYESPT=EYE SPOT (Kabatiella zeae or Aureobasidium zeae). A 1 to 9 visualrating indicating the resistance to Eye Spot. A higher score indicates ahigher resistance. Data are collected only when sufficient selectionpressure exists in the experiment measured.

FUSERS=FUSARIUM EAR ROT SCORE (Fusarum moniliforme or Fusariumsubglutinans). A 1 to 9 visual rating indicating the resistance toFusarium Ear Rot. A higher score indicates a higher resistance. Data arecollected only when sufficient selection pressure exists in theexperiment measured.

GDU=GROWING DEGREE UNITS. Using the Barger Heat Unit Theory, whichassumes that maize growth occurs in the temperature range 50 degrees F.−86 degrees F. and that temperatures outside this range slow downgrowth; the maximum daily heat unit accumulation is 36 and the minimumdaily heat unit accumulation is 0. The seasonal accumulation of GDU is amajor factor in determining maturity zones.

GDUSHD=GDU TO SHED. The number of growing degree units (GDUs) or heatunits required for an inbred line or hybrid to have approximately 50percent of the plants shedding pollen and is measured from the time ofplanting. Growing degree units are calculated by the Barger Method,where the heat units for a 24-hour period are:

${GDU} = {\frac{\left( {{Max}.{\mspace{11mu}\;}{temp}.{+ {{Min}.{\mspace{11mu}\;}{temp}.}}} \right)}{2} - 50}$

The highest maximum temperature used is 86 degrees F. and the lowestminimum temperature used is 50 degrees F. For each inbred or hybrid ittakes a certain number of GDUs to reach various stages of plantdevelopment.

GDUSLK=GDU TO SILK. The number of growing degree units required for aninbred line or hybrid to have approximately 50 percent of the plantswith silk emergence from time of planting. Growing degree units arecalculated by the Barger Method as given in GDU SHD definition.

GENE SILENCING. The interruption or suppression of the expression of agene at the level of transcription or translation.

GENOTYPE. Refers to the genetic constitution of a cell or organism.

GIBERS=GIBBERELLA EAR ROT (PINK MOLD) (Gibberella zeae). A 1 to 9 visualrating indicating the resistance to Gibberella Ear Rot. A higher scoreindicates a higher resistance. Data are collected only when sufficientselection pressure exists in the experiment measured.

GIBROT=GIBBERELLA STALK ROT SCORE. Score of stalk rot severity due toGibberella (Gibberella zeae). Expressed as a 1 to 9 score with 9 beinghighly resistant. Data are collected only when sufficient selectionpressure exists in the experiment measured.

GLFSPT=GRAY LEAF SPOT (Cercospora zeae-maydis). A 1 to 9 visual ratingindicating the resistance to Gray Leaf Spot. A higher score indicates ahigher resistance. Data are collected only when sufficient selectionpressure exists in the experiment measured.

GOSWLT=GOSS' WILT (Corynebacterium nebraskense). A 1 to 9 visual ratingindicating the resistance to Goss' Wilt. A higher score indicates ahigher resistance. Data are collected only when sufficient selectionpressure exists in the experiment measured.

GRNAPP=GRAIN APPEARANCE. This is a 1 to 9 rating for the generalappearance of the shelled grain as it is harvested based on such factorsas the color of harvested grain, any mold on the grain, and any crackedgrain. High scores indicate good grain visual quality.

HAPLOID PLANT PART. Refers to a plant part or cell that has the samehaploid genotype as PHE67.

HCBLT=HELMINTHOSPORIUM CARBONUM LEAF BLIGHT (Helminthosporum carbonum).A 1 to 9 visual rating indicating the resistance to Helminthosporiuminfection. A higher score indicates a higher resistance. Data arecollected only when sufficient selection pressure exists in theexperiment measured.

HD SMT=HEAD SMUT (Sphacelotheca reiliana). This score indicates thepercentage of plants not infected. Data are collected only whensufficient selection pressure exists in the experiment measured.

HSKCVR=HUSK COVER. A 1 to 9 score based on performance relative to keychecks, with a score of 1 indicating very short husks, tip of ear andkernels showing; 5 is intermediate coverage of the ear under mostconditions, sometimes with thin husk; and a 9 has husks extending andclosed beyond the tip of the ear. Scoring can best be done nearphysiological maturity stage or any time during dry down untilharvested.

INBRED. A line developed through inbreeding or doubled haploidy thatpreferably comprises homozygous alleles at about 95% or more of itsloci.

INC D/A=GROSS INCOME (DOLLARS PER ACRE). Relative income per acreassuming drying costs of two cents per point above 15.5 percent harvestmoisture and current market price per bushel.

INCOME/ACRE. Income advantage of hybrid to be patented over other hybridon per acre basis.

INC ADV=GROSS INCOME ADVANTAGE. Gross income advantage of variety #1over variety #2.

INTROGRESSION. The process of transferring genetic material from onegenotype to another.

KSZDCD=KERNEL SIZE DISCARD. The percent of discard seed; calculated asthe sum of discarded tip kernels and extra large kernels.

LINKAGE. Refers to a phenomenon wherein alleles on the same chromosometend to segregate together more often than expected by chance if theirtransmission was independent.

LINKAGE DISEQUILIBRIUM. Refers to a phenomenon wherein alleles tend toremain together in linkage groups when segregating from parents tooffspring, with a greater frequency than expected from their individualfrequencies.

LOCUS. A defined segment of DNA.

L/POP=YIELD AT LOW DENSITY. Yield ability at relatively low plantdensities on a 1 to 9 relative system with a higher number indicatingthe hybrid responds well to low plant densities for yield relative toother hybrids. A 1, 5, and 9 would represent very poor, average, andvery good yield response, respectively, to low plant density.

LRTLDG=LATE ROOT LODGING. The percentage of plants that do not rootlodge after anthesis through harvest; plants that lean from the verticalaxis at an approximately 30 degree angle or greater would be counted asroot lodged. Data are collected only when sufficient selection pressureexists in the experiment measured.

LRTLPN=LATE ROOT LODGING. An estimate of the percentage of plants thatdo not root lodge after anthesis through harvest; plants that lean fromthe vertical axis at an approximately 30 degree angle or greater wouldbe considered as root lodged. Data are collected only when sufficientselection pressure exists in the experiment measured.

LRTLSC=LATE ROOT LODGING SCORE. Score for severity of plants that leanfrom a vertical axis at an approximate 30 degree angle or greater whichtypically results from strong winds after flowering. Recorded prior toharvest when a root-lodging event has occurred. This lodging results inplants that are leaned or “lodged” over at the base of the plant and donot straighten or “goose-neck” back to a vertical position. Expressed asa 1 to 9 score with 9 being no lodging. Data are collected only whensufficient selection pressure exists in the experiment measured.

MDMCPX=MAIZE DWARF MOSAIC COMPLEX (MDMV=Maize Dwarf Mosaic Virus andMCDV=Maize Chlorotic Dwarf Virus). A 1 to 9 visual rating indicating theresistance to Maize Dwarf Mosaic Complex. A higher score indicates ahigher resistance. Data are collected only when sufficient selectionpressure exists in the experiment measured.

MST=HARVEST MOISTURE. The moisture is the actual percentage moisture ofthe grain at harvest.

MSTADV=MOISTURE ADVANTAGE. The moisture advantage of variety #1 overvariety #2 as calculated by: MOISTURE of variety #2—MOISTURE of variety#1=MOISTURE ADVANTAGE of variety #1.

NEI DISTANCE. A quantitative measure of percent similarity between twolines. Nei's distance between lines A and B can be defined as1-(2*number alleles in common/(number alleles in A+number alleles in B).For example, if lines A and B are the same for 95 out of 100 alleles,the Nei distance would be 0.05. If lines A and B are the same for 98 outof 100 alleles, the Nei distance would be 0.02. Free software forcalculating Nei distance is available on the internet at multiplelocations such as, for example, at:evolution.genetics.washington.edu/phylip.html. See Nei, Proc Natl AcadSci, 76:5269-5273 (1979) which is incorporated by reference for thispurpose.

NLFBLT=NORTHERN LEAF BLIGHT (Helminthosporium turcicum or Exserohilumturcicum). A 1 to 9 visual rating indicating the resistance to NorthernLeaf Blight. A higher score indicates a higher resistance. Data arecollected only when sufficient selection pressure exists in theexperiment measured.

OILT=GRAIN OIL. Absolute value of oil content of the kernel as predictedby Near-Infrared Transmittance and expressed as a percent of dry matter.

PEDIGREE DISTANCE. Relationship among generations based on theirancestral links as evidenced in pedigrees. May be measured by thedistance of the pedigree from a given starting point in the ancestry.

PERCENT IDENTITY. Percent identity as used herein refers to thecomparison of the homozygous alleles of two inbred lines. Each inbredplant will have the same allele (and therefore be homozygous) at almostall of their loci. Percent identity is determined by comparing astatistically significant number of the homozygous alleles of two inbredlines. For example, a percent identity of 90% between inbred PHE67 andother inbred line means that the two inbred lines have the same alleleat 90% of their loci.

PERCENT SIMILARITY. Percent similarity as used herein refers to thecomparison of the homozygous alleles of an inbred line with anotherplant. The homozygous alleles of PHE67 are compared with the alleles ofa non-inbred plant, such as a hybrid, and if the allele of the inbredmatches at least one of the alleles from the hybrid then they are scoredas similar. Percent similarity is determined by comparing astatistically significant number of loci and recording the number ofloci with similar alleles as a percentage. For example, a percentsimilarity of 90% between inbred PHE67 and a hybrid maize plant meansthat the inbred line matches at least one of the hybrid alleles at 90%of the loci. In the case of a hybrid produced from PHE67 as the male orfemale parent, such hybrid will comprise two sets of alleles, one set ofwhich will comprise the same alleles as the homozygous alleles of inbredline PHE67.

PLANT. As used herein, the term “plant” includes reference to animmature or mature whole plant, including a plant that has beendetasseled or from which seed or grain has been removed. Seed or embryothat will produce the plant is also considered to be the plant.

PLANT PARTS. As used herein, the term “plant parts” includes leaves,stems, roots, seed, grain, embryo, pollen, ovules, flowers, ears, cobs,husks, stalks, root tips, anthers, pericarp, silk, tissue, cells and thelike.

PLTHT=PLANT HEIGHT. This is a measure of the height of the plant fromthe ground to the tip of the tassel in centimeters.

POLSC=POLLEN SCORE. A 1 to 9 visual rating indicating the amount ofpollen shed. The higher the score the more pollen shed.

POLWT=POLLEN WEIGHT. This is calculated by dry weight of tasselscollected as shedding commences minus dry weight from similar tasselsharvested after shedding is complete.

POP K/A=PLANT POPULATIONS. Measured as 1000s per acre.

POP ADV=PLANT POPULATION ADVANTAGE. The plant population advantage ofvariety #1 over variety #2 as calculated by PLANT POPULATION of variety#2—PLANT POPULATION of variety #1=PLANT POPULATION ADVANTAGE of variety#1.

PRM=PREDICTED RELATIVE MATURITY. This trait, predicted relativematurity, is based on the harvest moisture of the grain. The relativematurity rating is based on a known set of checks and utilizes standardlinear regression analyses and is also referred to as the ComparativeRelative Maturity Rating System that is similar to the MinnesotaRelative Maturity Rating System.

PRMSHD=A relative measure of the growing degree units (GDU) required toreach 50% pollen shed. Relative values are predicted values from thelinear regression of observed GDU's on relative maturity of commercialchecks.

PROT=GRAIN PROTEIN. Absolute value of protein content of the kernel aspredicted by Near-infrared Transmittance and expressed as a percent ofdry matter.

RESISTANCE. Synonymous with tolerance. The ability of a plant towithstand exposure to an insect, disease, herbicide or other condition.A resistant plant variety will have a level of resistance higher than acomparable wild-type variety.

RTLDG=ROOT LODGING. Root lodging is the percentage of plants that do notroot lodge; plants that lean from the vertical axis at an approximately30 degree angle or greater would be counted as root lodged. Data arecollected only when sufficient selection pressure exists in theexperiment measured.

RTLADV=ROOT LODGING ADVANTAGE. The root lodging advantage of variety #1over variety #2. Data are collected only when sufficient selectionpressure exists in the experiment measured.

SCTGRN=SCATTER GRAIN. A 1 to 9 visual rating indicating the amount ofscatter grain (lack of pollination or kernel abortion) on the ear. Thehigher the score the less scatter grain.

SDGVGR=SEEDLING VIGOR. This is the visual rating (1 to 9) of the amountof vegetative growth after emergence at the seedling stage(approximately five leaves). A higher score indicates better vigor.

SEL IND=SELECTION INDEX. The selection index gives a single measure ofthe hybrid's worth based on information for up to five traits. A maizebreeder may utilize his or her own set of traits for the selectionindex. One of the traits that is almost always included is yield. Theselection index data presented in the tables represent the mean valueaveraged across testing stations.

SELF POLLINATION. A plant is self-pollinated if pollen from one floweris transferred to the same or another flower of the same plant.

SIB POLLINATION. A plant is sib-pollinated when individuals within thesame family or line are used for pollination.

SINGLE LOCUS CONVERSION TRAIT. A trait that can be introgressed into acorn line through introgression and/or transformation of a single locus.Examples of such single locus traits include mutant genes, transgenesand native traits finely mapped to a single locus. One or more singlelocus conversion traits may be introduced into a single corn line.

SLFBLT=SOUTHERN LEAF BLIGHT (Helminthosporium maydis or Bipolarismaydis). A 1 to 9 visual rating indicating the resistance to SouthernLeaf Blight. A higher score indicates a higher resistance. Data arecollected only when sufficient selection pressure exists in theexperiment measured.

SOURST=SOUTHERN RUST (Puccinia polysora). A 1 to 9 visual ratingindicating the resistance to Southern Rust. A higher score indicates ahigher resistance. Data are collected only when sufficient selectionpressure exists in the experiment measured.

STAGRN=STAY GREEN. Stay green is the measure of plant health near thetime of black layer formation (physiological maturity). A high scoreindicates better late-season plant health.

STDADV=STALK STANDING ADVANTAGE. The advantage of variety #1 overvariety #2 for the trait STK CNT.

STKCNT=NUMBER OF PLANTS. This is the final stand or number of plants perplot.

STKLDG=STALK LODGING REGULAR. This is the percentage of plants that didnot stalk lodge (stalk breakage) at regular harvest (when grain moistureis between about 20 and 30%) as measured by either natural lodging orpushing the stalks and determining the percentage of plants that breakbelow the ear. Data are collected only when sufficient selectionpressure exists in the experiment measured.

STKLDS=STALK LODGING SCORE. A plant is considered as stalk lodged if thestalk is broken or crimped between the ear and the ground. This can becaused by any or a combination of the following: strong winds late inthe season, disease pressure within the stalks, ECB damage orgenetically weak stalks. This trait should be taken just prior to or atharvest. Expressed on a 1 to 9 scale with 9 being no lodging. Data arecollected only when sufficient selection pressure exists in theexperiment measured.

STLLPN=LATE STALK LODGING. This is the percent of plants that did notstalk lodge (stalk breakage or crimping) at or around late seasonharvest (when grain moisture is below 20%) as measured by either naturallodging or pushing the stalks and determining the percentage of plantsthat break or crimp below the ear. Data are collected only whensufficient selection pressure exists in the experiment measured.

STLPCN=STALK LODGING REGULAR. This is an estimate of the percentage ofplants that did not stalk lodge (stalk breakage) at regular harvest(when grain moisture is between about 20 and 30%) as measured by eithernatural lodging or pushing the stalks and determining the percentage ofplants that break below the ear. Data are collected only when sufficientselection pressure exists in the experiment measured.

STRT=GRAIN STARCH. Absolute value of starch content of the kernel aspredicted by Near-infrared Transmittance and expressed as a percent ofdry matter.

STWWLT=Stewart's Wilt (Erwinia stewartii). A 1 to 9 visual ratingindicating the resistance to Stewart's Wilt. A higher score indicates ahigher resistance. Data are collected only when sufficient selectionpressure exists in the experiment measured.

TASBLS=TASSEL BLAST. A 1 to 9 visual rating was used to measure thedegree of blasting (necrosis due to heat stress) of the tassel at thetime of flowering. A 1 would indicate a very high level of blasting attime of flowering, while a 9 would have no tassel blasting. Data arecollected only when sufficient selection pressure exists in theexperiment measured.

TASSZ=TASSEL SIZE. A 1 to 9 visual rating was used to indicate therelative size of the tassel. The higher the rating the larger thetassel.

TAS WT=TASSEL WEIGHT. This is the average weight of a tassel (grams)just prior to pollen shed.

TEXEAR=EAR TEXTURE. A 1 to 9 visual rating was used to indicate therelative hardness (smoothness of crown) of mature grain. A 1 would bevery soft (extreme dent) while a 9 would be very hard (flinty or verysmooth crown).

TILLER=TILLERS. A count of the number of tillers per plot that couldpossibly shed pollen was taken. Data are given as a percentage oftillers: number of tillers per plot divided by number of plants perplot.

TSTWT=TEST WEIGHT (UNADJUSTED). The measure of the weight of the grainin pounds for a given volume (bushel).

TSWADV=TEST WEIGHT ADVANTAGE. The test weight advantage of variety #1over variety #2.

WIN M %=PERCENT MOISTURE WINS.

WIN Y %=PERCENT YIELD WINS.

YIELD BU/A=YIELD (BUSHELS/ACRE). Yield of the grain at harvest by weightor volume (bushels) per unit area (acre) adjusted to 15% moisture.

YLDADV=YIELD ADVANTAGE. The yield advantage of variety #1 over variety#2 as calculated by: YIELD of variety #1—YIELD variety #2=YIELDADVANTAGE of variety #1.

YLDSC=YIELD SCORE. A 1 to 9 visual rating was used to give a relativerating for yield based on plot ear piles. The higher the rating thegreater visual yield appearance.

Definitions for Area of Adaptability

When referring to area of adaptability, such term is used to describethe location with the environmental conditions that would be well suitedfor this maize line. Area of adaptability is based on a number offactors, for example: days to maturity, insect resistance, diseaseresistance, and drought resistance. Area of adaptability does notindicate that the maize line will grow in every location within the areaof adaptability or that it will not grow outside the area.

Central Corn Belt: Iowa, Illinois, Indiana

Drylands: non-irrigated areas of North Dakota, South Dakota, Nebraska,Kansas, Colorado, and Oklahoma

Eastern U.S.: Ohio, Pennsylvania, Delaware, Maryland, Virginia, and WestVirginia

North central U.S.: Minnesota and Wisconsin

Northeast: Michigan, New York, Vermont, and Ontario and Quebec Canada

Northwest U.S.: North Dakota, South Dakota, Wyoming, Washington, Oregon,Montana, Utah, and Idaho

South central U.S.: Missouri, Tennessee, Kentucky, Arkansas

Southeast U.S.: North Carolina, South Carolina, Georgia, Florida,Alabama, Mississippi, and Louisiana

Southwest U.S.: Texas, Oklahoma, New Mexico, Arizona

Western U.S.: Nebraska, Kansas, Colorado, and California

Maritime Europe: Northern France, Germany, Belgium, Netherlands andAustria

DETAILED DESCRIPTION OF THE INVENTION AND FURTHER EMBODIMENTS

All tables discussed in the Detailed Description of the Invention andFurther Embodiments section can found at the end of the section.

Morphological and Physiological Characteristics of PHE67

Inbred maize line PHE67 is a yellow flint-dent maize inbred that may beused as either a male or female in the production of the firstgeneration F1 maize hybrid although PHE67 may be best suited for use asa female. Inbred maize line PHE67 is best adapted to the Central CornBelt, Northwest, Northcentral, Northeast, Eastern, Western, and Drylandareas of U.S. and Ontario, Canada and can be used to produce hybridswith approximately 100 to 108 maturity based on the Comparative RelativeMaturity Rating System. Inbred maize line PHE67 demonstrates goodNorthern Corn Leaf Blight resistance, good early stand count and goodearly growth scores as an inbred per se. In hybrid combination, inbredPHE67 demonstrates above average grain yield potential, high grain testweight and low ear placement.

The inbred has shown uniformity and stability within the limits ofenvironmental influence for all the traits as described in the VarietyDescription Information (Table 1, found at the end of the section). Theinbred has been self-pollinated and ear-rowed a sufficient number ofgenerations with careful attention paid to uniformity of plant type toensure the homozygosity and phenotypic stability necessary for use incommercial hybrid seed production. The line has been increased both byhand and in isolated fields with continued observation for uniformity.No variant traits have been observed or are expected in PHE67.

Inbred maize line PHE67, being substantially homozygous, can bereproduced by planting seeds of the line, growing the resulting maizeplants under self-pollinating or sib-pollinating conditions withadequate isolation, and harvesting the resulting seed using techniquesfamiliar to the agricultural arts.

Genotypic Characteristics of PHE67

In addition to phenotypic observations, a plant can also be identifiedby its genotype. The genotype of a plant can be characterized through agenetic marker profile, which can identify plants of the same variety ora related variety, or be used to determine or validate a pedigree. TheSSR profile of Inbred PHE67 can be found in Table 2 at the end of thissection.

As a result of inbreeding, PHE67 is substantially homozygous. Thishomozygosity has been characterized at the loci shown in the markerprofile provided herein. An F1 hybrid made with PHE67 would comprise themarker profile of PHE67 shown herein. This is because an F1 hybrid isthe sum of its inbred parents, e.g., if one inbred parent is homozygousfor allele x at a particular locus, and the other inbred parent ishomozygous for allele y at that locus, the F1 hybrid will be x.y(heterozygous) at that locus. The profile can therefore be used toidentify hybrids comprising PHE67 as a parent, since such hybrids willcomprise two sets of alleles, one set of which will be from PHE67. Thedetermination of the male set of alleles and the female set of allelesmay be made by profiling the hybrid and the pericarp of the hybrid seed,which is composed of maternal parent cells. One way to obtain thepaternal parent profile is to subtract the pericarp profile from thehybrid profile.

Subsequent generations of progeny produced by selection and breeding areexpected to be of genotype x (homozygous), y (homozygous), or x.y(heterozygous) for these locus positions. When the F1 plant is used toproduce an inbred, the resulting inbred should be either x or y for thatallele. In that regard, a unique allele or combination of alleles uniqueto that inbred can be used to identify progeny plants that retain thoseunique alleles or combinations of alleles.

Therefore, in accordance with the above, an embodiment of this inventionis a PHE67 progeny maize plant or plant part that is a first generation(F1) hybrid maize plant comprising two sets of alleles, wherein one setof the alleles is the same as PHE67 at all of the SSR loci listed inTable 2. A maize cell wherein one set of the alleles is the same asPHE67 at all of the SSR loci listed in Table 2 is also an embodiment ofthe invention. This maize cell may be a part of a hybrid seed, plant orplant part produced by crossing PHE67 with another inbred maize plant.

Genetic marker profiles can be obtained by techniques such asRestriction Fragment Length Polymorphisms (RFLPs), Randomly AmplifiedPolymorphic DNAs (RAPDs), Arbitrarily Primed Polymerase Chain Reaction(AP-PCR), DNA Amplification Fingerprinting (DAF), Sequence CharacterizedAmplified Regions (SCARs), Amplified Fragment Length Polymorphisms(AFLPs), Simple Sequence Repeats (SSRs) which are also referred to asMicrosatellites, and Single Nucleotide Polymorphisms (SNPs). Forexample, see Berry, Don et al., “Assessing Probability of Ancestry UsingSimple Sequence Repeat Profiles: Applications to Maize Hybrids andInbreds”, Genetics, 2002, 161:813-824, and Berry, Don et al., “AssessingProbability of Ancestry Using Simple Sequence Repeat Profiles:Applications to Maize Inbred Lines and Soybean Varieties”, Genetics,2003,165: 331-342.

Particular markers used for these purposes are not limited to the set ofmarkers disclosed herein, but may include any type of marker and markerprofile which provides a means of distinguishing varieties. In additionto being used for identification of Inbred maize line PHE67, a hybridproduced through the use of PHE67, and the identification orverification of pedigree for progeny plants produced through the use ofPHE67, the genetic marker profile is also useful in further breeding andin developing an introgressed trait conversion of PHE67.

Means of performing genetic marker profiles using SSR polymorphisms arewell known in the art. SSRs are genetic markers based on polymorphismsin repeated nucleotide sequences, such as microsatellites. A markersystem based on SSRs can be highly informative in linkage analysisrelative to other marker systems in that multiple alleles may bepresent. Another advantage of this type of marker is that, through useof flanking primers, detection of SSRs can be achieved, for example, bythe polymerase chain reaction (PCR), thereby eliminating the need forlabor-intensive Southern hybridization. The PCR detection is done by useof two oligonucleotide primers flanking the polymorphic segment ofrepetitive DNA. Repeated cycles of heat denaturation of the DNA followedby annealing of the primers to their complementary sequences at lowtemperatures, and extension of the annealed primers with DNA polymerase,comprise the major part of the methodology.

Following amplification, markers can be scored by electrophoresis of theamplification products. Scoring of marker genotype is based on the sizeof the amplified fragment, which may be measured by the number of basepairs of the fragment. While variation in the primer used or inlaboratory procedures can affect the reported fragment size, relativevalues should remain constant regardless of the specific primer orlaboratory used. When comparing lines it is preferable if all SSRprofiles are performed in the same lab. The SSR analyses reported hereinwere conducted in-house at Pioneer Hi-Bred. An SSR service is availableto the public on a contractual basis by DNA Landmarks inSaint-Jean-sur-Richelieu, Quebec, Canada.

Primers used for the SSRs reported herein are publicly available and maybe found in the Maize GDB on the World Wide Web at maizegdb.org(sponsored by the USDA Agricultural Research Service), in Sharopova etal. (Plant Mol. Biol. 48(5-6):463-481), Lee et al. (Plant Mol. Biol.48(5-6); 453-461), or may be constructed from sequences if reportedherein. Primers may be constructed from publicly available sequenceinformation. Some marker information may also be available from DNALandmarks.

Map information is provided by bin number as reported in the Maize GDBfor the IBM 2 and/or IBM 2 Neighbors maps. The bin number digits to theleft of decimal point represent the chromosome on which such marker islocated, and the digits to the right of the decimal represent thelocation on such chromosome. A bin number.xx designation indicates thatthe bin location on that chromosome is not known. Map positions are alsoavailable on the Maize GDB for a variety of different mappingpopulations.

PHE67 and its plant parts can be identified through a molecular markerprofile. An inbred corn plant cell having the SSR genetic marker profileshown in Table 2 is an embodiment of the invention. Such plant cell maybe either diploid or haploid.

Also encompassed within the scope of the invention are plants and plantparts substantially benefiting from the use of PHE67 in theirdevelopment, such as PHE67 comprising a introgressed trait throughbackcross conversion or transformation, and which may be identified byhaving an SSR molecular marker profile with a high percent identity toPHE67, such as at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% identity.Likewise, percent similarity at these percentages may be used toidentify hybrid and other non-inbred plants produced by the use ofPHE67.

An embodiment of this invention is an inbred PHE67 progeny maize plantor plant part comprising the same homozygous alleles as the plant orplant part of PHE67 for at least 90% of the SSR loci listed in Table 2.A plant cell comprising the same homozygous alleles as a plant cell ofPHE67 for at least 90% of the SSR loci listed in Table 2 is also anembodiment of this invention. In these specific embodiments, 90% mayalso be replaced by any integer or partial integer percent of 80% orgreater as listed above. One means of producing such a progeny plant,plant part or cell is through the backcrossing and/or transformationmethods described herein.

Similarly, an embodiment of this invention is a PHE67 progeny maizeplant or plant part comprising at least one allele per locus that is thesame allele as the plant or plant part of PHE67 for at least 90% of theSSR loci listed in Table 2. This progeny plant may be a hybrid. Aprogeny or hybrid plant cell wherein at least one allele per locus thatis the same allele as the plant cell PHE67 for at least 90% of the SSRloci listed in Table 2 is also a specific embodiment of this invention.In these specific embodiments, 90% may also be replaced by any integerpercent listed above. One means of producing such a progeny or hybridplant, plant part or cell is through the backcrossing and/ortransformation methods described herein.

In addition, the SSR profile of PHE67 also can be used to identifyessentially derived varieties and other progeny lines developed from theuse of PHE67, as well as cells and other plant parts thereof. Progenyplants and plant parts produced using PHE67 may be identified by havinga molecular marker profile of at least 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%,70%, 71%, 72%,73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5%genetic contribution from inbred line PHE67, as measured by eitherpercent identity or percent similarity.

Comparing PHE67 to Other Inbreds

A breeder uses various methods to help determine which plants should beselected from segregating populations and ultimately which inbred lineswill be used to develop hybrids for commercialization. In addition toknowledge of the germplasm and plant genetics, a part of the selectionprocess is dependent on experimental design coupled with the use ofstatistical analysis. Experimental design and statistical analysis areused to help determine which plants, which family of plants, and finallywhich inbred lines and hybrid combinations are significantly better ordifferent for one or more traits of interest. Experimental designmethods are used to assess error so that differences between two inbredlines or two hybrid lines can be more accurately evaluated. Statisticalanalysis includes the calculation of mean values, determination of thestatistical significance of the sources of variation, and thecalculation of the appropriate variance components. Either a five or aone percent significance level is customarily used to determine whethera difference that occurs for a given trait is real or due to theenvironment or experimental error. One of ordinary skill in the art ofplant breeding would know how to evaluate the traits of two plantvarieties to determine if there is no significant difference between thetwo traits expressed by those varieties. For example, see Fehr, Walt,Principles of Cultivar Development, p. 261-286 (1987). Mean trait valuesmay be used to determine whether trait differences are significant.Trait values should preferably be measured on plants grown under thesame environmental conditions, and environmental conditions should beappropriate for the traits or traits being evaluated. Sufficientselection pressure should be present for optimum measurement of traitsof interest such as herbicide, insect or disease resistance. Similarly,an introgressed trait conversion of PHE67 for resistance, such asherbicide resistance, should not be compared to PHE67 in the presence ofthe herbicide when comparing non-resistance related traits such as plantheight and yield.

In Table 3, data from traits and characteristics of inbred maize linePHE67 per se are given and compared to other maize inbred lines andhybrids. The following are the results of these comparisons. The resultsin Table 3 show inbred PHE67 has significantly different traits comparedto other maize inbred lines.

The results in Table 3A compare inbred PHE67 to inbred PH4GP. Theresults show inbred PHE67 has significantly different moisture, earlygrowth, resistance to Northern Leaf Blight and resistance to FusariumEar Rot compared to PH4GP.

The results in Table 3B compare inbred PHE67 to inbred PHCER. Theresults show inbred PHE67 differs significantly over multiple traitsincluding plant height, ear height and resistance to Anthracnose StalkRot when compared to inbred PHCER.

Development of Maize Hybrids Using PHE67

A single cross maize hybrid results from the cross of two inbred lines,each of which has a genotype that complements the genotype of the other.The hybrid progeny of the first generation is designated F1. In thedevelopment of commercial hybrids in a maize plant breeding program,only the F1 hybrid plants are sought. F1 hybrids are more vigorous thantheir inbred parents. This hybrid vigor, or heterosis, can be manifestedin many polygenic traits, including increased vegetative growth andincreased yield.

PHE67 may be used to produce hybrid maize. One such embodiment is themethod of crossing inbred maize line PHE67 with another maize plant,such as a different maize inbred line, to form a first generation F1hybrid seed. The first generation F1 hybrid seed, plant and plant partproduced by this method is an embodiment of the invention. The firstgeneration F1 seed, plant and plant part will comprise an essentiallycomplete set of the alleles of inbred line PHE67. One of ordinary skillin the art can utilize either breeder books or molecular methods toidentify a particular F1 hybrid plant produced using inbred line PHE67.Further, one of ordinary skill in the art may also produce F1 hybridswith transgenic, male sterile and/or backcross conversions of inbredline PHE67

The development of a maize hybrid in a maize plant breeding programinvolves three steps: (1) the selection of plants from various germplasmpools for initial breeding crosses; (2) the selfing of the selectedplants from the breeding crosses for several generations to produce aseries of inbred lines, such as PHE67, which, although different fromeach other, breed true and are highly uniform; and (3) crossing theselected inbred lines with different inbred lines to produce thehybrids. During the inbreeding process in maize, the vigor of the linesdecreases, and so one would not be likely to use PHE67 directly toproduce grain. However, vigor is restored when PHE67 is crossed to adifferent inbred line to produce a commercial F1 hybrid. An importantconsequence of the homozygosity and homogeneity of the inbred line isthat the hybrid between a defined pair of inbreds may be reproducedindefinitely as long as the homogeneity of the inbred parents ismaintained.

PHE67 may be used to produce a single cross hybrid, a double crosshybrid, or a three-way hybrid. A single cross hybrid is produced whentwo inbred lines are crossed to produce the F1 progeny. A double crosshybrid is produced from four inbred lines crossed in pairs (A×B and C×D)and then the two F1 hybrids are crossed again (A×B)×(C×D). A three-waycross hybrid is produced from three inbred lines where two of the inbredlines are crossed (A×B) and then the resulting F1 hybrid is crossed withthe third inbred (A×B)×C. In each case, pericarp tissue from the femaleparent will be a part of and protect the hybrid seed.

Combining Ability of PHE67

Combining ability of a line, as well as the performance of the line perse, is a factor in the selection of improved maize inbreds. Combiningability refers to a line's contribution as a parent when crossed withother lines to form hybrids. The hybrids formed for the purpose ofselecting superior lines may be referred to as test crosses, and includecomparisons to other hybrid varieties grown in the same environment(same cross, location and time of planting). One way of measuringcombining ability is by using values based in part on the overall meanof a number of test crosses weighted by number of experiment andlocation combinations in which the hybrid combinations occurs. The meanmay be adjusted to remove environmental effects and known geneticrelationships among the lines.

General combining ability provides an overall score for the inbred overa large number of test crosses. Specific combining ability providesinformation on hybrid combinations formed by PHE67 and a specific inbredparent. A line such as PHE67 which exhibits good general combiningability may be used in a large number of hybrid combinations.

A general combining ability report for inbred PHE67 is provided in Table4. This data represents the overall mean value for these traits overhundreds of test crosses. Table 4 demonstrates that inbred PHE67 showsgood general combining ability for hybrid production.

Hybrid Comparisons

These hybrid comparisons represent specific hybrid crosses with PHE67and a comparison of these specific hybrids with other hybrids withfavorable characteristics. These comparisons illustrate the goodspecific combining ability of PHE67.

The results in Table 5 compare a specific hybrid for which inbred PHE67is a parent with other hybrids. The results show that inbred PHE67 showsgood specific combining ability.

The data in Table 6 show that numerous species of the genus of F1hybrids created with PHE67 have been reduced to practice. Phenotypicdata are presented for these hybrids and are based on replicated fieldtrials. Of course, many more species of this genus may be created by oneof ordinary skill in the art without undue experimentation by crossingPHE67 with a multitude of publicly available inbred lines. For example,see J. T. Gerdes et al., Compilation of North American Maize BreedingGermplasm, pp. 1-87 (Crop Science Society of America, 1993) which isincorporated by reference for this purpose.

Introgression of a New Locus or Trait into PHE67

PHE67 represents a new base genetic line into which a new locus or traitmay be introgressed. Direct transformation and backcrossing representtwo important methods that can be used to accomplish such anintrogression. The term backcross conversion and single locus conversionare used interchangeably to designate the product of a backcrossingprogram.

Backcross Conversions of PHE67

A backcross conversion of PHE67 occurs when DNA sequences are introducedthrough backcrossing (Hallauer et al. in Corn and Corn Improvement,Sprague and Dudley, Third Ed. 1998), with PHE67 utilized as therecurrent parent. Both naturally occurring and transgenic DNA sequencesmay be introduced through backcrossing techniques. A backcrossconversion may produce a plant with a trait or locus conversion in atleast one or more backcrosses, including at least 2 crosses, at least 3crosses, at least 4 crosses, at least 5 crosses and the like. Molecularmarker assisted breeding or selection may be utilized to reduce thenumber of backcrosses necessary to achieve the backcross conversion. Forexample, see Openshaw, S. J. et al., Marker-assisted Selection inBackcross Breeding, In: Proceedings Symposium of the Analysis ofMolecular Data, August, 1994, Crop Science Society of America,Corvallis, Oreg., where it is demonstrated that a backcross conversioncan be made in as few as two backcrosses.

The complexity of the backcross conversion method depends on the type oftrait being transferred (single genes or closely linked genes as vs.unlinked genes), the level of expression of the trait, the type ofinheritance (cytoplasmic or nuclear) and the types of parents includedin the cross. It is understood by those of ordinary skill in the artthat for single gene traits that are relatively easy to classify, thebackcross method is effective and relatively easy to manage. (SeeHallauer et al. in Corn and Corn Improvement, Sprague and Dudley, ThirdEd. 1998). Desired traits that may be transferred through backcrossconversion include, but are not limited to, waxy starch, sterility(nuclear and cytoplasmic), fertility restoration, grain color (white),nutritional enhancements, drought resistance, enhanced nitrogenutilization efficiency, altered nitrogen responsiveness, altered fattyacid profile, increased digestibility, low phytate, industrialenhancements, disease resistance (bacterial, fungal or viral), insectresistance, herbicide resistance and yield enhancements. In addition, anintrogression site itself, such as an FRT site, Lox site or other sitespecific integration site, may be inserted by backcrossing and utilizedfor direct insertion of one or more genes of interest into a specificplant variety. In some embodiments of the invention, the number of locithat may be backcrossed into PHE67 is at least 1, 2, 3, 4, or 5 and/orno more than 6, 5, 4, 3, or 2. A single loci may contain severaltransgenes, such as a transgene for disease resistance that, in the sameexpression vector, also contains a transgene for herbicide resistance.The gene for herbicide resistance may be used as a selectable markerand/or as a phenotypic trait. A single locus conversion of site specificintegration system allows for the integration of multiple genes at theconverted loci. Further, SSI and FRT technologies known to those ofskill in the art in the art may result in multiple gene introgressionsat a single locus.

The backcross conversion may result from either the transfer of adominant allele or a recessive allele. Selection of progeny containingthe trait of interest is accomplished by direct selection for a traitassociated with a dominant allele. Transgenes transferred viabackcrossing typically function as a dominant single gene trait and arerelatively easy to classify. Selection of progeny for a trait that istransferred via a recessive allele, such as the waxy starchcharacteristic, requires growing and selfing the first backcrossgeneration to determine which plants carry the recessive alleles.Recessive traits may require additional progeny testing in successivebackcross generations to determine the presence of the locus ofinterest. The last backcross generation is usually selfed to give purebreeding progeny for the gene(s) being transferred, although a backcrossconversion with a stably introgressed trait may also be maintained byfurther backcrossing to the recurrent parent with selection for theconverted trait.

Along with selection for the trait of interest, progeny are selected forthe phenotype of the recurrent parent. While occasionally additionalpolynucleotide sequences or genes may be transferred along with thebackcross conversion, the backcross conversion line “fits into the samehybrid combination as the recurrent parent inbred line and contributesthe effect of the additional gene added through the backcross.” Poehlmanet al. (1995, page 334). It has been proposed that in general thereshould be at least four backcrosses when it is important that therecovered lines be essentially identical to the recurrent parent exceptfor the characteristic being transferred (Fehr 1987, Principles ofCultivar Development). However, as noted above, the number ofbackcrosses necessary can be reduced with the use of molecular markers.Other factors, such as a genetically similar donor parent, may alsoreduce the number of backcrosses necessary.

One process for adding or modifying a trait or locus in maize inbredline PHE67 comprises crossing PHE67 plants grown from PHE67 seed withplants of another maize line that comprise the desired trait or locus,selecting F1 progeny plants that comprise the desired trait or locus toproduce selected F1 progeny plants, crossing the selected progeny plantswith the PHE67 plants to produce backcross progeny plants, selecting forbackcross progeny plants that have the desired trait or locus and themorphological characteristics of maize inbred line PHE67 to produceselected backcross progeny plants; and backcrossing to PHE67 three ormore times in succession to produce selected fourth or higher backcrossprogeny plants that comprise said trait or locus. The modified PHE67 maybe further characterized as having the physiological and morphologicalcharacteristics of maize inbred line PHE67 listed in Table 1 asdetermined at the 5% significance level when grown in the sameenvironmental conditions and/or may be characterized by percentsimilarity or identity to PHE67 as determined by SSR markers. The abovemethod may be utilized with fewer backcrosses in appropriate situations,such as when the donor parent is highly related or markers are used inthe selection step. Desired traits that may be used include thosenucleic acids known in the art, some of which are listed herein, thatwill affect traits through nucleic acid expression or inhibition.Desired loci include the introgression of FRT, Lox and other sites forsite specific integration.

In addition, the above process and other similar processes describedherein may be used to produce F1 hybrid maize seed by adding a step atthe end of the process that comprises crossing PHE67 with theintrogressed trait or locus with a different maize plant and harvestingthe resultant F1 hybrid maize seed.

Male Sterility and Hybrid Seed Production

Hybrid seed production requires elimination or inactivation of pollenproduced by the female inbred parent. Incomplete removal or inactivationof the pollen provides the potential for self-pollination. A reliablemethod of controlling male fertility in plants offers the opportunityfor improved seed production.

PHE67 can be produced in a male-sterile form. There are several ways inwhich a maize plant can be manipulated so that it is male sterile. Theseinclude use of manual or mechanical emasculation (or detasseling), useof one or more genetic factors that confer male sterility, includingcytoplasmic genetic and/or nuclear genetic male sterility, use ofgametocides and the like. A male sterile inbred designated PHE67 mayinclude one or more genetic factors, which result in cytoplasmic geneticand/or nuclear genetic male sterility. All of such embodiments arewithin the scope of the present claims. The male sterility may be eitherpartial or complete male sterility.

Hybrid maize seed is often produced by a male sterility systemincorporating manual or mechanical detasseling. Alternate strips of twomaize inbreds are planted in a field, and the pollen-bearing tassels areremoved from one of the inbreds (female). Provided that there issufficient isolation from sources of foreign maize pollen, the ears ofthe detasseled inbred will be fertilized only from the other inbred(male), and the resulting seed is therefore hybrid and will form hybridplants.

The laborious detasseling process can be avoided by using cytoplasmicmale-sterile (CMS) inbreds. Plants of a CMS inbred are male sterile as aresult of genetic factors in the cytoplasm, as opposed to the nucleus,and so nuclear linked genes are not transferred during backcrossing.Thus, this characteristic is inherited exclusively through the femaleparent in maize plants, since only the female provides cytoplasm to thefertilized seed. CMS plants are fertilized with pollen from anotherinbred that is not male-sterile. Pollen from the second inbred may ormay not contribute genes that make the hybrid plants male-fertile, andeither option may be preferred depending on the intended use of thehybrid. The same hybrid seed, a portion produced from detasseled fertilemaize and a portion produced using the CMS system, can be blended toinsure that adequate pollen loads are available for fertilization whenthe hybrid plants are grown. CMS systems have been successfully usedsince the 1950's, and the male sterility trait is routinely backcrossedinto inbred lines. See Wych, p. 585-586,1998.

There are several methods of conferring genetic male sterilityavailable, such as multiple mutant genes at separate locations withinthe genome that confer male sterility, as disclosed in U.S. Pat. Nos.4,654,465 and 4,727,219 to Brar et al. and chromosomal translocations asdescribed by Patterson in U.S. Pat. Nos. 3,861,709 and 3,710,511. Inaddition to these methods, Albertsen et al., U.S. Pat. No. 5,432,068,describe a system of nuclear male sterility which includes: identifyinga gene which is critical to male fertility; silencing this native genewhich is critical to male fertility; removing the native promoter fromthe essential male fertility gene and replacing it with an induciblepromoter; inserting this geneticsally engineered gene back into theplant; and thus creating a plant that is male sterile because theinducible promoter is not “on” resulting in the male fertility gene notbeing transcribed. Fertility is restored by inducing, or turning “on”,the promoter, which in turn allows the gene that confers male fertilityto be transcribed.

These, and the other methods of conferring genetic male sterility in theart, each possess their own benefits and drawbacks. Some other methodsuse a variety of approaches such as delivering into the plant a geneencoding a cytotoxic substance associated with a male tissue specificpromoter or an antisense system in which a gene critical to fertility isidentified and an antisense to that gene is inserted in the plant (seeFabinjanski, et al. EPO 89/3010153.8 publication no. 329,308 and PCTapplication PCT/CA90/00037 published as WO 90/08828).

Another system for controlling male sterility makes use of gametocides.Gametocides are not a genetic system, but rather a topical applicationof chemicals. These chemicals affect cells that are critical to malefertility. The application of these chemicals affects fertility in theplants only for the growing season in which the gametocide is applied(see Carlson, Glenn R., U.S. Pat. No.: 4,936,904). Application of thegametocide, timing of the application and genotype specificity oftenlimit the usefulness of the approach and it is not appropriate in allsituations.

Incomplete control over male fertility may result in self-pollinatedseed being unintentionally harvested and packaged with hybrid seed. Thiswould typically be only female parent seed, because the male plant isgrown in rows that are typically destroyed prior to seed development.Once the seed from the hybrid bag is planted, it is possible to identifyand select these self-pollinated plants. These self-pollinated plantswill be one of the inbred lines used to produce the hybrid. Though thepossibility of inbred PHE67 being included in a hybrid seed bag exists,the occurrence is very low because much care is taken by seed companiesto avoid such inclusions. It is worth noting that hybrid seed is sold togrowers for the production of grain or forage and not for breeding orseed production. These self-pollinated plants can be identified andselected by one skilled in the art due to their less vigorous appearancefor vegetative and/or reproductive characteristics, including shorterplant height, small ear size, ear and kernel shape, cob color, or othercharacteristics.

Identification of these self-pollinated lines can also be accomplishedthrough molecular marker analyses. See, “The Identification of FemaleSelfs in Hybrid Maize: A Comparison Using Electrophoresis andMorphology”, Smith, J. S. C. and Wych, R. D., Seed Science andTechnology 14, 1-8 (1995), the disclosure of which is expresslyincorporated herein by reference. Through these technologies, thehomozygosity of the self pollinated line can be verified by analyzingallelic composition at various loci along the genome. Those methodsallow for rapid identification of the invention disclosed herein. Seealso, “Identification of Atypical Plants in Hybrid Maize Seed byPostcontrol and Electrophoresis” Sarca, V. et al., Probleme de GeneticaTeoritica si Aplicata Vol. 20 (1) p. 29-42.

An embodiment of this invention is a process for producing seed of PHE67comprising planting a collection of seed comprising seed of a hybrid,one of whose parents is inbred PHE67 said collection also comprisingseed of said inbred, growing plants from said collection of seed,identifying inbred parent plants, selecting said inbred parent plant;and controlling pollination to preserve the homozygosity of said inbredparent plant.

Transformation

The advent of new molecular biological techniques has allowed theisolation and characterization of genetic elements with specificfunctions, such as encoding specific protein products. Scientists in thefield of plant biology developed a strong interest in engineering thegenome of plants to contain and express foreign genetic elements, oradditional, or modified versions of native or endogenous geneticelements in order to alter the traits of a plant in a specific manner.Any DNA sequences, whether from a different species or from the samespecies, which are inserted into the genome using transformation arereferred to herein collectively as “transgenes”. In some embodiments ofthe invention, a transformed variant of PHE67 may contain at least onetransgene but could contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10and/or no more than 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2.Over the last fifteen to twenty years several methods for producingtransgenic plants have been developed, and the present invention alsorelates to transformed versions of the claimed inbred maize line PHE67as well as hybrid combinations thereof.

Numerous methods for plant transformation have been developed, includingbiological and physical plant transformation protocols. See, forexample, Miki et al., “Procedures for Introducing Foreign DNA intoPlants” in Methods in Plant Molecular Biology and Biotechnology, Glick,B. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993) pages67-88 and Armstrong, “The First Decade of Maize Transformation: A Reviewand Future Perspective” (Maydica 44:101-109, 1999). In addition,expression vectors and in vitro culture methods for plant cell or tissuetransformation and regeneration of plants are available. See, forexample, Gruber et al., “Vectors for Plant Transformation” in Methods inPlant Molecular Biology and Biotechnology, Glick, B. R. and Thompson, J.E. Eds. (CRC Press, Inc., Boca Raton, 1993) pages 89-119.

The most prevalent types of plant transformation involve theconstruction of an expression vector. Such a vector comprises a DNAsequence that contains a gene under the control of or operatively linkedto a regulatory element, for example a promoter. The vector may containone or more genes and one or more regulatory elements.

A genetic trait which has been engineered into the genome of aparticular maize plant using transformation techniques, could be movedinto the genome of another line using traditional breeding techniquesthat are well known in the plant breeding arts. For example, abackcrossing approach is commonly used to move a transgene from atransformed maize plant to an elite inbred line, and the resultingprogeny would then comprise the transgene(s). Also, if an inbred linewas used for the transformation then the transgenic plants could becrossed to a different inbred in order to produce a transgenic hybridmaize plant.

Various genetic elements can be introduced into the plant genome usingtransformation. These elements include, but are not limited to genes;coding sequences; inducible, constitutive, and tissue specificpromoters; enhancing sequences; and signal and targeting sequences. Forexample, see the traits, genes and transformation methods listed in U.S.Pat. Nos. 6,118,055 and 6,284,953, which are herein incorporated byreference. In addition, transformability of a line can be increased byintrogressing the trait of high transformability from another line knownto have high transformability, such as Hi-II. See U.S. PatentApplication Publication US2004/0016030 (2004).

With transgenic plants according to the present invention, a foreignprotein can be produced in commercial quantities. Thus, techniques forthe selection and propagation of transformed plants, which are wellunderstood in the art, yield a plurality of transgenic plants that areharvested in a conventional manner, and a foreign protein then can beextracted from a tissue of interest or from total biomass. Proteinextraction from plant biomass can be accomplished by known methods whichare discussed, for example, by Heney and Orr, Anal. Biochem. 114: 92-6(1981).

A genetic map can be generated, primarily via conventional RestrictionFragment Length Polymorphisms (RFLP), Polymerase Chain Reaction (PCR)analysis, Simple Sequence Repeats (SSR) and Single NucleotidePolymorphisms (SNP) that identifies the approximate chromosomal locationof the integrated DNA molecule. For exemplary methodologies in thisregard, see Glick and Thompson, Methods in Plant Molecular Biology andBiotechnology 269-284 (CRC Press, Boca Raton, 1993).

Wang et al. discuss “Large Scale Identification, Mapping and Genotypingof Single-Nucleotide Polymorphisms in the Human Genome”, Science,280:1077-1082, 1998, and similar capabilities are available for the corngenome. Map information concerning chromosomal location is useful forproprietary protection of a subject transgenic plant. If unauthorizedpropagation is undertaken and crosses made with other germplasm, the mapof the integration region can be compared to similar maps for suspectplants to determine if the latter have a common parentage with thesubject plant. Map comparisons would involve hybridizations, RFLP, PCR,SSR and sequencing, all of which are conventional techniques. SNPs mayalso be used alone or in combination with other techniques.

Likewise, by means of the present invention, plants can be geneticallyengineered to express various phenotypes of agronomic interest. Throughthe transformation of maize the expression of genes can be altered toenhance disease resistance, insect resistance, herbicide resistance,agronomic traits, grain quality and other traits. Transformation canalso be used to insert DNA sequences which control or help controlmale-sterility. DNA sequences native to maize as well as non-native DNAsequences can be transformed into maize and used to alter levels ofnative or non-native proteins. Various promoters, targeting sequences,enhancing sequences, and other DNA sequences can be inserted into themaize genome for the purpose of altering the expression of proteins.Reduction of the activity of specific genes (also known as genesilencing, or gene suppression) is desirable for several aspects ofgenetic engineering in plants.

Many techniques for gene silencing are well known to one of skill in theart, including but not limited to knock-outs (such as by insertion of atransposable element such as mu (Vicki Chandler, The Maize Handbook ch.118 (Springer-Verlag 1994) or other genetic elements such as a FRT, Loxor other site specific integration site, antisense technology (see,e.g., Sheehy et al. (1988) PNAS USA 85:8805-8809; and U.S. Pat. Nos.5,107,065; 5,453,566; and 5,759,829); co-suppression (e.g., Taylor(1997) Plant Cell 9:1245; Jorgensen (1990) Trends Biotech.8(12):340-344; Flavell (1994) PNAS USA 91:3490-3496; Finnegan et al.(1994) Bio/Technology 12: 883-888; and Neuhuber et al. (1994) Mol. Gen.Genet. 244:230-241); RNA interference (Napoli et al. (1990) Plant Cell2:279-289; U.S. Pat. No. 5,034,323; Sharp (1999) Genes Dev. 13:139-141;Zamore et al. (2000) Cell 101:25-33; and Montgomery et al. (1998) PNASUSA 95:15502-15507), virus-induced gene silencing (Burton, et al. (2000)Plant Cell 12:691-705; and Baulcombe (1999) Curr. Op. Plant Bio.2:109-113); target-RNA-specific ribozymes (Haseloff et al. (1988) Nature334: 585-591); hairpin structures (Smith et al. (2000) Nature407:319-320; WO 99/53050; and WO 98/53083); MicroRNA (Aukerman & Sakai(2003) Plant Cell 15:2730-2741); ribozymes (Steinecke et al. (1992) EMBOJ. 11:1525; and Perriman et al. (1993) Antisense Res. Dev. 3:253);oligonucleotide mediated targeted modification (e.g., WO 03/076574 andWO 99/25853); Zn-finger targeted molecules (e.g., WO 01/52620; WO03/048345; and WO 00/42219); and other methods or combinations of theabove methods known to those of skill in the art.

Exemplary nucleotide sequences that may be altered by geneticengineering include, but are not limited to, those categorized below.

1. Transgenes that Confer Resistance to Insects or Disease and thatEncode:

(A) Plant disease resistance genes. Plant defenses are often activatedby specific interaction between the product of a disease resistance gene(R) in the plant and the product of a corresponding avirulence (Avr)gene in the pathogen. A plant variety can be transformed with clonedresistance gene to engineer plants that are resistant to specificpathogen strains. See, for example Jones et al., Science 266: 789 (1994)(cloning of the tomato Cf-9 gene for resistance to Cladosporium fulvum);Martin et al., Science 262: 1432 (1993) (tomato Pto gene for resistanceto Pseudomonas syringae pv. tomato encodes a protein kinase); Mindrinoset al., Cell 78: 1089 (1994) (Arabidopsis RSP2 gene for resistance toPseudomonas syringae); McDowell & Woffenden, (2003) Trends Biotechnol.21(4): 178-83 and Toyoda et al., (2002) Transgenic Res. 11(6):567-82. Aplant resistant to a disease is one that is more resistant to a pathogenas compared to the wild type plant.

(B) A Bacillus thuringiensis protein, a derivative thereof or asynthetic polypeptide modeled thereon. See, for example, Geiser et al.,Gene 48: 109 (1986), who disclose the cloning and nucleotide sequence ofa Bt delta-endotoxin gene. Moreover, DNA molecules encodingdelta-endotoxin genes can be purchased from American Type CultureCollection (Rockville, Md.), for example, under ATCC Accession Nos.40098, 67136, 31995 and 31998. Other examples of Bacillus thuringiensistransgenes being genetically engineered are given in the followingpatents and patent applications and hereby are incorporated by referencefor this purpose: U.S. Pat. Nos. 5,188,960; 5,689,052; 5,880,275; WO91/14778; WO 99/31248; WO 01/12731; WO 99/24581; WO 97/40162 and U.S.application Ser. Nos. 10/032,717; 10/414,637; and 10/606,320.

(C) An insect-specific hormone or pheromone such as an ecdysteroid andjuvenile hormone, a variant thereof, a mimetic based thereon, or anantagonist or agonist thereof. See, for example, the disclosure byHammock et al., Nature 344: 458 (1990), of baculovirus expression ofcloned juvenile hormone esterase, an inactivator of juvenile hormone.

(D) An insect-specific peptide which, upon expression, disrupts thephysiology of the affected pest. For example, see the disclosures ofRegan, J. Biol. Chem. 269: 9 (1994) (expression cloning yields DNAcoding for insect diuretic hormone receptor); Pratt et al., Biochem.Biophys. Res. Comm. 163: 1243 (1989) (an allostatin is identified inDiploptera puntata); Chattopadhyay et al. (2004) Critical Reviews inMicrobiology 30 (1): 33-54 2004; Zjawiony (2004) J Nat Prod 67 (2):300-310; Carlini & Grossi-de-Sa (2002) Toxicon, 40 (11): 1515-1539;Ussuf et al. (2001) Curr Sci. 80 (7): 847-853; and Vasconcelos &Oliveira (2004) Toxicon 44 (4): 385-403. See also U.S. Pat. No.5,266,317 to Tomalski et al., who disclose genes encodinginsect-specific toxins.

(E) An enzyme responsible for a hyperaccumulation of a monterpene, asesquiterpene, a steroid, hydroxamic acid, a phenylpropanoid derivativeor another non-protein molecule with insecticidal activity.

(F) An enzyme involved in the modification, including thepost-translational modification, of a biologically active molecule; forexample, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme,a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, aphosphatase, a kinase, a phosphorylase, a polymerase, an elastase, achitinase and a glucanase, whether natural or synthetic. See PCTapplication WO 93/02197 in the name of Scott et al., which discloses thenucleotide sequence of a callase gene. DNA molecules which containchitinase-encoding sequences can be obtained, for example, from the ATCCunder Accession Nos. 39637 and 67152. See also Kramer et al., InsectBiochem. Molec. Biol. 23: 691 (1993), who teach the nucleotide sequenceof a cDNA encoding tobacco hookworm chitinase, and Kawalleck et al.,Plant Molec. Biol. 21: 673 (1993), who provide the nucleotide sequenceof the parsley ubi4-2 polyubiquitin gene, U.S. application Ser. Nos.10/389,432, 10/692,367, and U.S. Pat. No. 6,563,020.

(G) A molecule that stimulates signal transduction. For example, see thedisclosure by Botella et al., Plant Molec. Biol. 24: 757 (1994), ofnucleotide sequences for mung bean calmodulin cDNA clones, and Griess etal., Plant Physiol. 104: 1467 (1994), who provide the nucleotidesequence of a maize calmodulin cDNA clone.

(H) A hydrophobic moment peptide. See PCT application WO 95/16776 andU.S. Pat. No. 5,580,852 (disclosure of peptide derivatives ofTachyplesin which inhibit fungal plant pathogens) and PCT application WO95/18855 and U.S. Pat. No. 5,607,914) (teaches synthetic antimicrobialpeptides that confer disease resistance).

(I) A membrane permease, a channel former or a channel blocker. Forexample, see the disclosure by Jaynes et al., Plant Sci. 89: 43 (1993),of heterologous expression of a cecropin-beta lytic peptide analog torender transgenic tobacco plants resistant to Pseudomonas solanacearum.

(J) A viral-invasive protein or a complex toxin derived therefrom. Forexample, the accumulation of viral coat proteins in transformed plantcells imparts resistance to viral infection and/or disease developmenteffected by the virus from which the coat protein gene is derived, aswell as by related viruses. See Beachy et al., Ann. Rev. Phytopathol.28: 451 (1990). Coat protein-mediated resistance has been conferred upontransformed plants against alfalfa mosaic virus, cucumber mosaic virus,tobacco streak virus, potato virus X, potato virus Y, tobacco etchvirus, tobacco rattle virus and tobacco mosaic virus. Id.

(K) An insect-specific antibody or an immunotoxin derived therefrom.Thus, an antibody targeted to a critical metabolic function in theinsect gut would inactivate an affected enzyme, killing the insect. Cf.Taylor et al., Abstract #497, SEVENTH INT'L SYMPOSIUM ON MOLECULARPLANT-MICROBE INTERACTIONS (Edinburgh, Scotland, 1994) (enzymaticinactivation in transgenic tobacco via production of single-chainantibody fragments).

(L) A virus-specific antibody. See, for example, Tavladoraki et al.,Nature 366: 469 (1993), who show that transgenic plants expressingrecombinant antibody genes are protected from virus attack.

(M) A developmental-arrestive protein produced in nature by a pathogenor a parasite. Thus, fungal endo alpha-1,4-D-polygalacturonasesfacilitate fungal colonization and plant nutrient release bysolubilizing plant cell wall homo-alpha-1,4-D-galacturonase. See Lamb etal., Bio/Technology 10: 1436 (1992). The cloning and characterization ofa gene which encodes a bean endopolygalacturonase-inhibiting protein isdescribed by Toubart et al., Plant J. 2: 367 (1992).

(N) A developmental-arrestive protein produced in nature by a plant. Forexample, Logemann et al., Bio/Technology 10: 305 (1992), have shown thattransgenic plants expressing the barley ribosome-inactivating gene havean increased resistance to fungal disease.

(O) Genes involved in the Systemic Acquired Resistance (SAR) Responseand/or the pathogenesis related genes. Briggs, S., Current Biology,5(2):128-131 (1995), Pieterse &Van Loon (2004) Curr. Opin. Plant Bio.7(4):456-64 and Somssich (2003) Cell 113(7):815-6.

(P) Antifungal genes (Cornelissen and Melchers, PI. Physiol.101:709-712, (1993) and Parijs et al., Planta 183:258-264, (1991) andBushnell et al., Can. J. of Plant Path. 20(2):137-149 (1998). Also seeU.S. application Ser. No. 09/950,933.

(Q) Detoxification genes, such as for fumonisin, beauvericin,moniliformin and zearalenone and their structurally related derivatives.For example, see U.S. Pat. No. 5,792,931.

(R) Cystatin and cysteine proteinase inhibitors. See U.S. applicationSer. No: 10/947,979.

(S) Defensin genes. See WO03000863 and U.S. application Ser. No.10/178,213.

(T) Genes conferring resistance to nematodes. See WO 03/033651 and Urwinet. al., Planta 204:472479 (1998), Williamson (1999) Curr Opin PlantBio. 2(4):327-31.

(U) Genes such as rcg1conferring resistance to Anthracnose stalk rot,which is caused by the fungus Colletotrichum graminiola. See M. Jung etal., Generation-means analysis and quantitative trait locus mapping ofAnthracnose Stalk Rot genes in Maize, Theor. Appl. Genet. (1994)89:413-418 which is incorporated by reference for this purpose, as wellas U.S. Patent Application 60/675,664, which is also incorporated byreference for this purpose.

2. Transgenes that Confer Resistance to a Herbicide, for Example:

(A) A herbicide that inhibits the growing point or meristem, such as animidazolinone or a sulfonylurea. Exemplary genes in this category codefor mutant ALS and AHAS enzyme as described, for example, by Lee et al.,EMBO J. 7:1241 (1988), and Miki et al., Theor. Appl. Genet. 80: 449(1990), respectively. See also, U.S. Pat. Nos. 5,605,011; 5,013,659;5,141,870; 5,767,361; 5,731,180; 5,304,732; 4,761,373; 5,331,107;5,928,937; and 5,378,824; and international publication WO 96/33270,which are incorporated herein by reference for this purpose.

(B) Glyphosate (resistance imparted by mutant5-enolpyruvl-3-phosphikimate synthase (EPSP) and aroA genes,respectively) and other phosphono compounds such as glufosinate(phosphinothricin acetyl transferase (PAT) and Streptomyceshygroscopicus phosphinothricin acetyl transferase (bar) genes), andpyridinoxy or phenoxy proprionic acids and cycloshexones (ACCaseinhibitor-encoding genes). See, for example, U.S. Pat. No. 4,940,835 toShah et al., which discloses the nucleotide sequence of a form of EPSPSwhich can confer glyphosate resistance. U.S. Pat. No. 5,627,061 to Barryet al. also describes genes encoding EPSPS enzymes. See also U.S. Pat.Nos. 6,566,587; 6,338,961; 6,248,876 B1; 6,040,497; 5,804,425;5,633,435; 5,145,783; 4,971,908; 5,312,910; 5,188,642; 4,940,835;5,866,775; 6,225,114 B1; 6,130,366; 5,310,667; 4,535,060; 4,769,061;5,633,448; 5,510,471; Re. 36,449; RE 37,287 E; and 5,491,288; andinternational publications EP1173580; WO 01/66704; EP1173581 andEP1173582, which are incorporated herein by reference for this purpose.Glyphosate resistance is also imparted to plants that express a genethat encodes a glyphosate oxido-reductase enzyme as described more fullyin U.S. Pat. Nos. 5,776,760 and 5,463,175, which are incorporated hereinby reference for this purpose. In addition glyphosate resistance can beimparted to plants by the over expression of genes encoding glyphosateN-acetyltransferase. See, for example, U.S. application Ser. Nos.US01/46227; 10/427,692 and 10/427,692. A DNA molecule encoding a mutantaroA gene can be obtained under ATCC accession No. 39256, and thenucleotide sequence of the mutant gene is disclosed in U.S. Pat. No.4,769,061 to Comai. European Patent Application No. 0 333 033 to Kumadaet al. and U.S. Pat. No. 4,975,374 to Goodman et al. disclose nucleotidesequences of glutamine synthetase genes which confer resistance toherbicides such as L-phosphinothricin. The nucleotide sequence of aphosphinothricin-acetyl-transferase gene is provided in European PatentNo. 0 242 246 and 0 242 236 to Leemans et al. De Greef et al.,Bio/Technology 7: 61 (1989), describe the production of transgenicplants that express chimeric bar genes coding for phosphinothricinacetyl transferase activity. See also, U.S. Pat. Nos. 5,969,213;5,489,520; 5,550,318; 5,874,265; 5,919,675; 5,561,236; 5,648,477;5,646,024; 6,177,616 B1; and 5,879,903, which are incorporated herein byreference for this purpose. Exemplary genes conferring resistance tophenoxy proprionic acids and cycloshexones, such as sethoxydim andhaloxyfop, are the Acc1-S1, Acc1-S2 and Acc1-S3 genes described byMarshall et al., Theor. Appl. Genet. 83: 435 (1992).

(C) A herbicide that inhibits photosynthesis, such as a triazine (psbAand gs+ genes) and a benzonitrile (nitrilase gene). Przibilla et al.,Plant Cell 3: 169 (1991), describe the transformation of Chlamydomonaswith plasmids encoding mutant psbA genes. Nucleotide sequences fornitrilase genes are disclosed in U.S. Pat. No. 4,810,648 to Stalker, andDNA molecules containing these genes are available under ATCC AccessionNos. 53435, 67441 and 67442. Cloning and expression of DNA coding for aglutathione S-transferase is described by Hayes et al., Biochem. J. 285:173 (1992).

(D) Acetohydroxy acid synthase, which has been found to make plants thatexpress this enzyme resistant to multiple types of herbicides, has beenintroduced into a variety of plants (see, e.g., Hattori et al. (1995)Mol Gen Genet 246:419). Other genes that confer resistance to herbicidesinclude: a gene encoding a chimeric protein of rat cytochrome P4507A1and yeast NADPH-cytochrome P450 oxidoreductase (Shiota et al. (1994)Plant Physiol. 106(1):17-23), genes for glutathione reductase andsuperoxide dismutase (Aono et al. (1995) Plant Cell Physiol 36:1687, andgenes for various phosphotransferases (Datta et al. (1992) Plant MolBiol 20:619).

(E) Protoporphyrinogen oxidase (protox) is necessary for the productionof chlorophyll, which is necessary for all plant survival. The protoxenzyme serves as the target for a variety of herbicidal compounds. Theseherbicides also inhibit growth of all the different species of plantspresent, causing their total destruction. The development of plantscontaining altered protox activity which are resistant to theseherbicides are described in U.S. Pat. Nos. 6,288,306 B1; 6,282,837 B1;and 5,767,373; and international publication WO 01/12825.

3. Transgenes that Confer or Contribute to an Altered GrainCharacteristic, Such As:

(A) Altered fatty acids, for example, by

-   -   (1) Down-regulation of stearoyl-ACP desaturase to increase        stearic acid content of the plant. See Knultzon et al., Proc.        Natl. Acad. Sci. USA 89: 2624 (1992) and WO99/64579 (Genes for        Desaturases to Alter Lipid Profiles in Corn),    -   (2) Elevating oleic acid via FAD-2 gene modification and/or        decreasing linolenic acid via FAD-3 gene modification (see U.S.        Pat. Nos. 6,063,947; 6,323,392; 6,372,965 and WO 93/11245),    -   (3) Altering conjugated linolenic or linoleic acid content, such        as in WO 01/12800,    -   (4) Altering LEC1, AGP, Dek1, Superal1,mi1ps, various Ipa genes        such as Ipa1, Ipa3, hpt or hggt. For example, see WO 02/42424,        WO 98/22604, WO 03/011015, U.S. Pat. No. 6,423,886, U.S. Pat.        No. 6,197,561, U.S. Pat. No. 6,825,397, US2003/0079247,        US2003/0204870, WO02/057439, WO03/011015 and Rivera-Madrid, R.        et. al. Proc. Natl. Acad. Sci. 92:5620-5624 (1995).

(B) Altered phosphorus content, for example, by the

-   -   (1) Introduction of a phytase-encoding gene would enhance        breakdown of phytate, adding more free phosphate to the        transformed plant. For example, see Van Hartingsveldt et al.,        Gene 127: 87 (1993), for a disclosure of the nucleotide sequence        of an Aspergillus niger phytase gene.    -   (2) Up-regulation of a gene that reduces phytate content. In        maize, this, for example, could be accomplished, by cloning and        then re-introducing DNA associated with one or more of the        alleles, such as the LPA alleles, identified in maize mutants        characterized by low levels of phytic acid, such as in Raboy et        al., Maydica 35:

383 (1990) and/or by altering inositol kinase activity as in WO02/059324, US2003/0009011, WO 03/027243, US2003/0079247, WO 99/05298,U.S. Pat. No. 6,197,561, U.S. Pat. No. 6,291,224, U.S. Pat. No.6,391,348, WO2002/059324, US2003/0079247, Wo98/45448, WO99/55882,WO01/04147.

(C) Altered carbohydrates effected, for example, by altering a gene foran enzyme that affects the branching pattern of starch or a genealtering thioredoxin such as NTR and/or TRX (see U.S. Pat. No. 6,531,648which is incorporated by reference for this purpose) and/or a gamma zeinknock out or mutant such as cs27 or TUSC27 or en27 (See U.S. Pat. No.6,858,778 and US2005/0160488, US2005/0204418; which are incorporated byreference for this purpose). See Shiroza et al., J. Bacteriol. 170: 810(1988) (nucleotide sequence of Streptococcus mutans fructosyltransferasegene), Steinmetz et al., Mol. Gen. Genet. 200: 220 (1985) (nucleotidesequence of Bacillus subtilis levansucrase gene), Pen et al.,Bio/Technology 10: 292 (1992) (production of transgenic plants thatexpress Bacillus licheniformis alpha-amylase), Elliot et al., PlantMolec. Biol. 21: 515 (1993) (nucleotide sequences of tomato invertasegenes), Søgaard et al., J. Biol. Chem. 268: 22480 (1993) (site-directedmutagenesis of barley alpha-amylase gene), and Fisher et al., PlantPhysiol. 102: 1045 (1993) (maize endosperm starch branching enzyme 11),WO 99/10498 (improved digestibility and/or starch extraction throughmodification of UDP-D-xylose 4-epimerase, Fragile 1 and 2, Refl, HCHL,C4H), U.S. Pat. No. 6,232,529 (method of producing high oil seed bymodification of starch levels (AGP)). The fatty acid modification genesmentioned above may also be used to affect starch content and/orcomposition through the interrelationship of the starch and oilpathways.

(D) Altered antioxidant content or composition, such as alteration oftocopherol or tocotrienols. For example, see U.S. Pat. No. 6,787,683,US2004/0034886 and WO 00/68393 involving the manipulation of antioxidantlevels through alteration of a phytl prenyl transferase (ppt), WO03/082899 through alteration of a homogentisate geranyl geranyltransferase (hggt).

(E) Altered essential seed amino acids. For example, see U.S. Pat. No.6,127,600 (method of increasing accumulation of essential amino acids inseeds), U.S. Pat. No. 6,080,913 (binary methods of increasingaccumulation of essential amino acids in seeds), U.S. Pat. No. 5,990,389(high lysine), WO99/40209 (alteration of amino acid compositions inseeds), WO99/29882 (methods for altering amino acid content ofproteins), U.S. Pat. No. 5,850,016 (alteration of amino acidcompositions in seeds), WO98/20133 (proteins with enhanced levels ofessential amino acids), U.S. Pat. No. 5,885,802 (high methionine), U.S.Pat. No. 5,885,801 (high threonine), U.S. Pat. No. 6,664,445 (plantamino acid biosynthetic enzymes), U.S. Pat. No. 6,459,019 (increasedlysine and threonine), U.S. Pat. No. 6,441,274 (plant tryptophansynthase beta subunit), U.S. Pat. No. 6,346,403 (methionine metabolicenzymes), U.S. Pat. No. 5,939,599 (high sulfur), U.S. Pat. No. 5,912,414(increased methionine), WO98/56935 (plant amino acid biosyntheticenzymes), WO98/45458 (engineered seed protein having higher percentageof essential amino acids), WO98/42831 (increased lysine), U.S. Pat. No.5,633,436 (increasing sulfur amino acid content), U.S. Pat. No.5,559,223 (synthetic storage proteins with defined structure containingprogrammable levels of essential amino acids for improvement of thenutritional value of plants), WO96/01905 (increased threonine),WO95/15392 (increased lysine), US2003/0163838, US2003/0150014,US2004/0068767, U.S. Pat. No. 6,803,498, WO01/79516, and WO00/09706 (CesA: cellulose synthase), U.S. Pat. No. 6,194,638 (hemicellulose), U.S.Pat. No. 6,399,859 and US2004/0025203 (UDPGdH), U.S. Pat. No. 6,194,638(RGP).

4. Genes that Control Male-Sterility

There are several methods of conferring genetic male sterilityavailable, such as multiple mutant genes at separate locations withinthe genome that confer male sterility, as disclosed in U.S. Pat. Nos.4,654,465 and 4,727,219 to Brar et al. and chromosomal translocations asdescribed by Patterson in U.S. Pat. Nos. 3,861,709 and 3,710,511. Inaddition to these methods, Albertsen et al., U.S. Pat. No. 5,432,068,describe a system of nuclear male sterility which includes: identifyinga gene which is critical to male fertility; silencing this native genewhich is critical to male fertility; removing the native promoter fromthe essential male fertility gene and replacing it with an induciblepromoter; inserting this genetically engineered gene back into theplant; and thus creating a plant that is male sterile because theinducible promoter is not “on” resulting in the male fertility gene notbeing transcribed. Fertility is restored by inducing, or turning “on”,the promoter, which in turn allows the gene that confers male fertilityto be transcribed.

(A) Introduction of a deacetylase gene under the control of atapetum-specific promoter and with the application of the chemicalN-Ac-PPT (WO 01/29237).

(B) Introduction of various stamen-specific promoters (WO 92/13956, WO92/13957).

(C) Introduction of the barnase and the barstar gene (Paul et al. PlantMol. Biol. 19:611-622, 1992).

For additional examples of nuclear male and female sterility systems andgenes, see also, U.S. Pat. Nos. 5,859,341; 6,297,426; 5,478,369;5,824,524; 5,850,014; and 6,265,640; all of which are herebyincorporated by reference.

5. Genes that create a site for site specific DNA integration. Thisincludes the introduction of FRT sites that may be used in the FLP/FRTsystem and/or Lox sites that may be used in the Cre/Loxp system. Forexample, see Lyznik, et al., Site-Specific Recombination for GeneticEngineering in Plants, Plant Cell Rep (2003) 21:925-932 and WO 99/25821,which are hereby incorporated by reference. Other systems that may beused include the Gin recombinase of phage Mu (Maeser et al., 1991; VickiChandler, The Maize Handbook ch. 118 (Springer-Verlag 1994), the Pinrecombinase of E. coli (Enomoto et al., 1983), and the R/RS system ofthe pSR1 plasmid (Araki et al., 1992).

6. Genes that affect abiotic stress resistance (including but notlimited to flowering, ear and seed development, enhancement of nitrogenutilization efficiency, altered nitrogen responsiveness, droughtresistance or tolerance, cold resistance or tolerance, and saltresistance or tolerance) and increased yield under stress. For example,see: WO 00/73475 where water use efficiency is altered throughalteration of malate; U.S. Pat. No. 5,892,009, U.S. Pat. No. 5,965,705,U.S. Pat. No. 5,929,305, U.S. Pat. No. 5,891,859, U.S. Pat. No.6,417,428, U.S. Pat. No. 6,664,446, U.S. Pat. No. 6,706,866, U.S. Pat.No. 6,717,034, WO2000060089, WO2001026459, WO2001035725, WO2001034726,WO2001035727, WO2001036444, WO2001036597, WO2001036598, WO2002015675,WO2002017430, WO2002077185, WO2002079403, WO2003013227, WO2003013228,WO2003014327, WO2004031349, WO2004076638, WO9809521, and WO9938977describing genes, including CBF genes and transcription factorseffective in mitigating the negative effects of freezing, high salinity,and drought on plants, as well as conferring other positive effects onplant phenotype; US2004/0148654 and WO01/36596 where abscisic acid isaltered in plants resulting in improved plant phenotype such asincreased yield and/or increased tolerance to abiotic stress;WO2000/006341, WO04/090143, U.S. application Ser. Nos. 10/817483 and09/545,334 where cytokinin expression is modified resulting in plantswith increased stress tolerance, such as drought tolerance, and/orincreased yield. Also see WO0202776, WO2003052063, JP2002281975, U.S.Pat. No. 6,084,153, WO0164898, U.S. Pat. No. 6,177,275, and U.S. Pat.No. 6,107,547 (enhancement of nitrogen utilization and altered nitrogenresponsiveness). For ethylene alteration, see US20040128719,US20030166197 and WO200032761. For plant transcription factors ortranscriptional regulators of abiotic stress, see e.g. US20040098764 orUS20040078852.

Other genes and transcription factors that affect plant growth andagronomic traits such as yield, flowering, plant growth and/or plantstructure, can be introduced or introgressed into plants, see e.g.WO97/49811 (LHY), WO98/56918 (ESD4), WO97/10339 and U.S. Pat. No.6,573,430 (TFL), U.S. Pat. No. 6,713,663 (FT), WO96/14414 (CON),WO96/38560, WO01/21822 (VRN1), WO00/44918 (VRN2), WO99/49064 (GI),WO00/46358 (FRI), WO97/29123, U.S. Pat. No. 6,794,560, U.S. Pat. No.6,307,126 (GAI), WO099/09174 (D8 and Rht), and WO2004076638 andWO2004031349 (transcription factors).

Using PHE67 to Develop Other Maize Inbreds

Inbred maize lines such as PHE67 are typically developed for use in theproduction of hybrid maize lines. However, inbred lines such as PHE67also provide a source of breeding material that may be used to developnew maize inbred lines. Plant breeding techniques known in the art andused in a maize plant breeding program include, but are not limited to,recurrent selection, mass selection, bulk selection, mass selection,backcrossing, pedigree breeding, open pollination breeding, restrictionfragment length polymorphism enhanced selection, genetic marker enhancedselection, making double haploids, and transformation. Oftencombinations of these techniques are used. The development of maizehybrids in a maize plant breeding program requires, in general, thedevelopment of homozygous inbred lines, the crossing of these lines, andthe evaluation of the crosses. There are many analytical methodsavailable to evaluate the result of a cross. The oldest and mosttraditional method of analysis is the observation of phenotypic traitsbut genotypic analysis may also be used.

Using PHE67 in a Breeding Program

This invention is directed to methods for producing a maize plant bycrossing a first parent maize plant with a second parent maize plantwherein either the first or second parent maize plant is an inbred maizeplant of the line PHE67. The other parent may be any other maize plant,such as another inbred line or a plant that is part of a synthetic ornatural population. Any such methods using the inbred maize line PHE67are part of this invention: selfing, sibbing, backcrosses, massselection, pedigree breeding, bulk selection, hybrid production, crossesto populations, and the like. These methods are well known in the artand some of the more commonly used breeding methods are described below.Descriptions of breeding methods can also be found in one of severalreference books (e.g., Allard, Principles of Plant Breeding, 1960;Simmonds, Principles of Crop Improvement, 1979; Fehr, “Breeding Methodsfor Cultivar Development”, Production and Uses, 2^(nd) ed., Wilcoxeditor, 1987 the disclosure of which is incorporated herein byreference).

Pedigree Breeding

Pedigree breeding starts with the crossing of two genotypes, such asPHE67 and one other elite inbred line having one or more desirablecharacteristics that is lacking or which complements PHE67. If the twooriginal parents do not provide all the desired characteristics, othersources can be included in the breeding population. In the pedigreemethod, superior plants are selfed and selected in successive filialgenerations. In the succeeding filial generations the heterozygouscondition gives way to homogeneous lines as a result of self-pollinationand selection. Typically in the pedigree method of breeding, five ormore successive filial generations of selfing and selection ispracticed: F1→F2; F2→F3; F3→F4; F4→F5, etc. After a sufficient amount ofinbreeding, successive filial generations will serve to increase seed ofthe developed inbred. Preferably, the inbred line comprises homozygousalleles at about 95% or more of its loci.

In addition to being used to create a backcross conversion, backcrossingcan also be used in combination with pedigree breeding to modify PHE67and a hybrid that is made using the modified PHE67. As discussedpreviously, backcrossing can be used to transfer one or morespecifically desirable traits from one line, the donor parent, to aninbred called the recurrent parent, which has overall good agronomiccharacteristics yet lacks that desirable trait or traits. However, thesame procedure can be used to move the progeny toward the genotype ofthe recurrent parent but at the same time retain many components of thenon-recurrent parent by stopping the backcrossing at an early stage andproceeding with selfing and selection. For example, an F1, such as acommercial hybrid, is created. This commercial hybrid may be backcrossedto one of its parent lines to create a BC1 or BC2. Progeny are selfedand selected so that the newly developed inbred has many of theattributes of the recurrent parent and yet several of the desiredattributes of the non-recurrent parent. This approach leverages thevalue and strengths of the recurrent parent for use in new hybrids andbreeding.

Therefore, an embodiment of this invention is a method of making abackcross conversion of maize inbred line PHE67, comprising the steps ofcrossing a plant of maize inbred line PHE67 with a donor plantcomprising a mutant gene or transgene conferring a desired trait,selecting an F1 progeny plant comprising the mutant gene or transgeneconferring the desired trait, and backcrossing the selected F1 progenyplant to a plant of maize inbred line PHE67. This method may furthercomprise the step of obtaining a molecular marker profile of maizeinbred line PHE67 and using the molecular marker profile to select for aprogeny plant with the desired trait and the molecular marker profile ofPHE67. In the same manner, this method may be used to produce an F1hybrid seed by adding a final step of crossing the desired traitconversion of maize inbred line PHE67 with a different maize plant tomake F1 hybrid maize seed comprising a mutant gene or transgeneconferring the desired trait.

Recurrent Selection and Mass Selection

Recurrent selection is a method used in a plant breeding program toimprove a population of plants. PHE67 is suitable for use in a recurrentselection program. The method entails individual plants crosspollinating with each other to form progeny. The progeny are grown andthe superior progeny selected by any number of selection methods, whichinclude individual plant, half-sib progeny, full-sib progeny, selfedprogeny and topcrossing. The selected progeny are cross pollinated witheach other to form progeny for another population. This population isplanted and again superior plants are selected to cross pollinate witheach other. Recurrent selection is a cyclical process and therefore canbe repeated as many times as desired. The objective of recurrentselection is to improve the traits of a population. The improvedpopulation can then be used as a source of breeding material to obtaininbred lines to be used in hybrids or used as parents for a syntheticcultivar. A synthetic cultivar is the resultant progeny formed by theintercrossing of several selected inbreds.

Mass selection is a useful technique when used in conjunction withmolecular marker enhanced selection. In mass selection seeds fromindividuals are selected based on phenotype and/or genotype. Theseselected seeds are then bulked and used to grow the next generation.Bulk selection requires growing a population of plants in a bulk plot,allowing the plants to self-pollinate, harvesting the seed in bulk andthen using a sample of the seed harvested in bulk to plant the nextgeneration. Instead of self pollination, directed pollination could beused as part of the breeding program.

Mutation Breeding

Mutation breeding is one of many methods that could be used to introducenew traits into PHE67. Mutations that occur spontaneously or areartificially induced can be useful sources of variability for a plantbreeder. The goal of artificial mutagenesis is to increase the rate ofmutation for a desired characteristic. Mutation rates can be increasedby many different means including temperature, long-term seed storage,tissue culture conditions, radiation; such as X-rays, Gamma rays (e.g.cobalt 60 or cesium 137), neutrons, (product of nuclear fission byuranium 235 in an atomic reactor), Beta radiation (emitted fromradioisotopes such as phosphorus 32 or carbon 14), or ultravioletradiation (preferably from 2500 to 2900 nm), or chemical mutagens (suchas base analogues (5-bromo-uracil), related compounds (8-ethoxycaffeine), antibiotics (streptonigrin), alkylating agents (sulfurmustards, nitrogen mustards, epoxides, ethylenamines, sulfates,sulfonates, sulfones, lactones), azide, hydroxylamine, nitrous acid, oracridines. Once a desired trait is observed through mutagenesis thetrait may then be incorporated into existing germplasm by traditionalbreeding techniques, such as backcrossing. Details of mutation breedingcan be found in “Principles of Cultivar Development” Fehr, 1993Macmillan Publishing Company, the disclosure of which is incorporatedherein by reference. In addition, mutations created in other lines maybe used to produce a backcross conversion of PHE67 that comprises suchmutation.

Breeding with Molecular Markers

Molecular markers, which includes markers identified through the use oftechniques such as Isozyme Electrophoresis, Restriction Fragment LengthPolymorphisms (RFLPs), Randomly Amplified Polymorphic DNAs (RAPDs),Arbitrarily Primed Polymerase Chain Reaction (AP-PCR), DNA AmplificationFingerprinting (DAF), Sequence Characterized Amplified Regions (SCARs),Amplified Fragment Length Polymorphisms (AFLPs), Simple Sequence Repeats(SSRs) and Single Nucleotide Polymorphisms (SNPs), may be used in plantbreeding methods utilizing PHE67.

Isozyme Electrophoresis and RFLPs as discussed in Lee, M., “Inbred Linesof Maize and Their Molecular Markers,” The Maize Handbook,(Springer-Verlag, New York, Inc. 1994, at 423-432), have been widelyused to determine genetic composition. Isozyme Electrophoresis has arelatively low number of available markers and a low number of allelicvariants among maize inbreds. RFLPs allow more discrimination becausethey have a higher degree of allelic variation in maize and a largernumber of markers can be found. Both of these methods have been eclipsedby SSRs as discussed in Smith et al., “An evaluation of the utility ofSSR loci as molecular markers in maize (Zea mays L.): comparisons withdata from RFLPs and pedigree”, Theoretical and Applied Genetics (1997)vol. 95 at 163-173 and by Pejic et al., “Comparative analysis of geneticsimilarity among maize inbreds detected by RFLPs, RAPDs, SSRs, andAFLPs,” Theoretical and Applied Genetics (1998) at 1248-1255incorporated herein by reference. SSR technology is more efficient andpractical to use than RFLPs; more marker loci can be routinely used andmore alleles per marker locus can be found using SSRs in comparison toRFLPs. Single Nucleotide Polymorphisms may also be used to identify theunique genetic composition of the invention and progeny lines retainingthat unique genetic composition. Various molecular marker techniques maybe used in combination to enhance overall resolution.

Maize DNA molecular marker linkage maps have been rapidly constructedand widely implemented in genetic studies. One such study is describedin Boppenmaier, et al., “Comparisons among strains of inbreds forRFLPs”, Maize Genetics Cooperative Newsletter, 65:1991, pg. 90, isincorporated herein by reference.

One use of molecular markers is Quantitative Trait Loci (QTL) mapping.QTL mapping is the use of markers, which are known to be closely linkedto alleles that have measurable effects on a quantitative trait.Selection in the breeding process is based upon the accumulation ofmarkers linked to the positive effecting alleles and/or the eliminationof the markers linked to the negative effecting alleles from the plant'sgenome.

Molecular markers can also be used during the breeding process for theselection of qualitative traits. For example, markers closely linked toalleles or markers containing sequences within the actual alleles ofinterest can be used to select plants that contain the alleles ofinterest during a backcrossing breeding program. The markers can also beused to select for the genome of the recurrent parent and against thegenome of the donor parent. Using this procedure can minimize the amountof genome from the donor parent that remains in the selected plants. Itcan also be used to reduce the number of crosses back to the recurrentparent needed in a backcrossing program. The use of molecular markers inthe selection process is often called genetic marker enhanced selection.

Production of Double Haploids

The production of double haploids can also be used for the developmentof inbreds in the breeding program. For example, an F1 hybrid for whichPHE67 is a parent can be used to produce double haploid plants. Doublehaploids are produced by the doubling of a set of chromosomes (1N) froma heterozygous plant to produce a completely homozygous individual. Forexample, see Wan et al., “Efficient Production of Doubled Haploid PlantsThrough Colchicine Treatment of Anther-Derived Maize Callus”,Theoretical and Applied Genetics, 77:889-892, 1989 and US2003/0005479.This can be advantageous because the process omits the generations ofselfing needed to obtain a homozygous plant from a heterozygous source.

Haploid induction systems have been developed for various plants toproduce haploid tissues, plants and seeds. The haploid induction systemcan produce haploid plants from any genotype by crossing a selected line(as female) with an inducer line. Such inducer lines for maize includeStock 6 (Coe, 1959, Am. Nat. 93:381-382; Sharkar and Coe, 1966, Genetics54:453-464) RWS (see world wide web sitewww.uni-hohenheim.de/%7Eipspwww/350b/indexe.html#Project3), KEMS(Deimling, Roeber, and Geiger, 1997, Vortr. Pflanzenzuchtg 38:203-224),or KMS and ZMS (Chalyk, Bylich & Chebotar, 1994, MNL 68:47; Chalyk &Chebotar, 2000, Plant Breeding 119:363-364), and indeterminategametophyte (ig) mutation (Kermicle 1969 Science 166:1422-1424). Thedisclosures of which are incorporated herein by reference.

Methods for obtaining haploid plants are also disclosed in Kobayashi, M.et al., Journ. of Heredity 71(1):9-14, 1980, Pollacsek, M., Agronomie(Paris) 12(3):247-251, 1992; Cho-Un-Haing et al., Journ. of Plant Biol.,1996, 39(3):185-188; Verdoodt, L., et al., February 1998, 96(2):294-300;Genetic Manipulation in Plant Breeding, Proceedings InternationalSymposium Organized by EUCARPIA, Sep. 8-13, 1985, Berlin, Germany;Chalyk et al., 1994, Maize Genet Coop. Newsletter 68:47; Chalyk, S. T.,1999, Maize Genet Coop. Newsletter 73:53-54; Coe, R. H., 1959, Am. Nat.93:381-382; Deimling, S. et al., 1997, Vortr. Pflanzenzuchtg 38:203-204;Kato, A., 1999, J. Hered. 90:276-280; Lashermes, P. et al., 1988, Theor.Appl. Genet. 76:570-572 and 76:405410; Tyrnov, V. S. et al., 1984, Dokl.Akad. Nauk. SSSR 276:735-738; Zabirova, E. R. et al., 1996, Kukuruza ISorgo N4, 17-19; Aman, M. A., 1978, Indian J. Genet Plant Breed38:452-457; Chalyk S. T., 1994, Euphytica 79:13-18; Chase, S. S., 1952,Agron. J. 44:263-267; Coe, E. H., 1959, Am. Nat. 93:381-382; Coe, E. H.,and Sarkar, K. R., 1964 J. Hered. 55:231-233; Greenblatt, I. M. andBock, M., 1967, J. Hered. 58:9-13; Kato, A., 1990, Maize Genet Coop.Newsletter 65:109-110; Kato, A., 1997, Sex. Plant Reprod. 10:96-100;Nanda, D. K. and Chase, S. S., 1966, Crop Sci. 6:213-215; Sarkar, K. R.and Coe, E. H., 1966, Genetics 54:453-464; Sarkar, K. R. and Coe, E. H.,1971, Crop Sci. 11:543-544; Sarkar, K. R. and Sachan J. K. S., 1972,Indian J. Agric. Sci. 42:781-786; Kermicle J. L., 1969, Mehta Yeshwant,M. R., Genetics and Molecular Biology, September 2000, 23(3):617-622;Tahir, M. S. et al. Pakistan Journal of Scientific and IndustrialResearch, August 2000, 43(4):258-261; Knox, R. E. et al. Plant Breeding,August 2000, 119(4):289-298; U.S. Pat. No. 5,639,951 and U.S. patentapplication Ser. No. 10/121,200, the disclosures of which areincorporated herein by reference.

Thus, an embodiment of this invention is a process for making asubstantially homozygous PHE67 progeny plant by producing or obtaining aseed from the cross of PHE67 and another maize plant and applying doublehaploid methods to the F1 seed or F1 plant or to any successive filialgeneration. Such methods decrease the number of generations required toproduce an inbred with similar genetics or characteristics to PHE67. SeeBernardo, R. and Kahler, A. L., Theor. Appl. Genet. 102:986-992, 2001.

In particular, a process of making seed retaining the molecular markerprofile of maize inbred line PHE67 is contemplated, such processcomprising obtaining or producing F1 hybrid seed for which maize inbredline PHE67 is a parent, inducing doubled haploids to create progenywithout the occurrence of meiotic segregation, obtaining the molecularmarker profile of maize inbred line PHE67, and selecting progeny thatretain the molecular marker profile of PHE67.

Use of PHE67 in Tissue Culture

This invention is also directed to the use of PHE67 in tissue culture.As used herein, the term “tissue culture” includes plant protoplasts,plant cell tissue culture, cultured microspores, plant calli, plantclumps, and the like. As used herein, phrases such as “growing the seed”or “grown from the seed” include embryo rescue, isolation of cells fromseed for use in tissue culture, as well as traditional growing methods.

Duncan, Williams, Zehr, and Widholm, Planta (1985) 165:322-332 reflectsthat 97% of the plants cultured that produced callus were capable ofplant regeneration. Subsequent experiments with both inbreds and hybridsproduced 91% regenerable callus that produced plants. In a further studyin 1988, Songstad, Duncan & Widholm in Plant Cell Reports (1988),7:262-265 reports several media additions that enhance regenerability ofcallus of two inbred lines. Other published reports also indicated that“nontraditional” tissues are capable of producing somatic embryogenesisand plant regeneration. K. P. Rao, et al., Maize Genetics CooperationNewsletter, 60:64-65 (1986), refers to somatic embryogenesis from glumecallus cultures and B. V. Conger, et al., Plant Cell Reports, 6:345-347(1987) indicates somatic embryogenesis from the tissue cultures of maizeleaf segments. Thus, it is clear from the literature that the state ofthe art is such that these methods of obtaining plants are, and were,“conventional” in the sense that they are routinely used and have a veryhigh rate of success.

Tissue culture of maize, including tassel/anther culture, is describedin U.S. 2002/0062506A1 and European Patent Application, publicationEP0160,390, each of which are incorporated herein by reference for thispurpose. Maize tissue culture procedures are also described in Green andRhodes, “Plant Regeneration in Tissue Culture of Maize,” Maize forBiological Research (Plant Molecular Biology Association,Charlottesville, Va. 1982, at 367-372) and in Duncan, et al., “TheProduction of Callus Capable of Plant Regeneration from Immature Embryosof Numerous Zea Mays Genotypes,” 165 Planta 322-332 (1985). Thus,another aspect of this invention is to provide cells which upon growthand differentiation produce maize plants having the genotype and/orphysiological and morphological characteristics of inbred line PHE67.

Progeny Plants

All plants produced by the use of the methods described herein and thatretain the unique genetic or trait combinations of PHE67 are within thescope of the invention. Progeny of the breeding methods described hereinmay be characterized in any number of ways, such as by traits retainedin the progeny, pedigree and/or molecular markers. Combinations of thesemethods of characterization may be used.

Breeder's of ordinary skill in the art have developed the concept of an“essentially derived variety”, which is defined in 7 U.S.C. § 2104(a)(3)of the Plant Variety Protection Act and is hereby incorporated byreference. Varieties and plants that are essentially derived from PHE67are within the scope of the invention.

Pedigree is a method used by breeders of ordinary skill in the art todescribe the varieties. Varieties that are more closely related bypedigree are likely to share common genotypes and combinations ofphenotypic characteristics. All breeders of ordinary skill in the artmaintain pedigree records of their breeding programs. These pedigreerecords contain a detailed description of the breeding process,including a listing of all parental lines used in the breeding processand information on how such line was used. One embodiment of thisinvention is progeny plants and parts thereof with at least one ancestorthat is PHE67, and more specifically, where the pedigree of the progenyincludes 1, 2, 3, 4, and/or 5 or less breeding crosses to a maize plantother than PHE67 or a plant that has PHE67 as a parent or otherprogenitor. A breeder of ordinary skill in the art would know if PHE67were used in the development of a progeny line, and would also know howmany crosses to a line other than PHE67 or line with PHE67 as a parentor other progenitor were made in the development of any progeny line.

Molecular markers also provide a means by which those of ordinary skillin the art characterize the similarity or differences of two lines.Using the breeding methods described herein, one can develop individualplants, plant cells, and populations of plants that retain at least 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%,68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 99.5% genetic contribution from inbred line PHE67,as measured by either percent identity or percent similarity. On average50% of the starting germplasm would be expected to be passed to theprogeny line after one cross to another line, 25% after another cross toa different line, and so on. With backcrossing, the expectedcontribution of PHE67 after 2, 3, 4 and 5 doses (or 1, 2, 3 and 4backcrosses) would be 75%, 87.5%, 93.75% and 96.875% respectively.Actual genetic contribution may be much higher than the geneticcontribution expected by pedigree, especially if molecular markers areused in selection. Molecular markers could also be used to confirmand/or determine the pedigree of the progeny line.

Traits are also used by those of ordinary skill in the art tocharacterize progeny. Traits are commonly evaluated at a significancelevel, such as a 1%, 5% or 10% significance level, when measured inplants grown in the same environmental conditions. For example, abackcross conversion of PHE67 may be characterized as having the samemorphological and physiological traits as PHE67. The traits used forcomparison may be those traits shown in Table 1, Table 3, Table 4 orTable 5.

A breeder will commonly work to combine a specific trait of anundeveloped variety of the species, such as a high level of resistanceto a particular disease, with one or more of the elite agronomiccharacteristics (yield, maturity, plant size, lodging resistance, etc.)needed for use as a commercial variety. This combination, oncedeveloped, provides a valuable source of new germplasm for furtherbreeding. For example, it may take 10-15 years and significant effort toproduce such a combination, yet progeny may be developed that retainthis combination in as little as 2-5 years and with much less effort.

Specific Embodiments

Specific methods and products produced using inbred line PHE67 in plantbreeding are discussed in the following sections. The methods outlinedare described in detail by way of illustration and example for purposesof clarity and understanding. However, it will be obvious that certainchanges and modifications may be practiced within the scope of theinvention.

One method for producing a line derived from inbred line PHE67 is asfollows. One of ordinary skill in the art would produce or obtain a seedfrom the cross between inbred line PHE67 and another variety of maize,such as an elite inbred variety. The F1 seed derived from this crosswould be grown to form a homogeneous population. The F1 seed wouldcontain essentially all of the alleles from variety PHE67 andessentially all of the alleles from the other maize variety. The F1nuclear genome would be made-up of 50% variety PHE67 and 50% of theother elite variety. The F1 seed would be grown and allowed to self,thereby forming F2 seed. On average the F2 seed would have derived 50%of its alleles from variety PHE67 and 50% from the other maize variety,but many individual plants from the population would have a greaterpercentage of their alleles derived from PHE67 (Wang J. and R. Bernardo,2000, Crop Sci. 40:659-665 and Bernardo, R. and A. L. Kahler, 2001,Theor. Appl. Genet 102:986-992). The molecular markers of PHE67 could beused to select and retain those lines with high similarity to PHE67. TheF2 seed would be grown and selection of plants would be made based onvisual observation, markers and/or measurement of traits. The traitsused for selection may be any PHE67 trait described in thisspecification, including the inbred per se maize PHE67 traits describedherein under the detailed description of inbred PHE67. Such traits mayalso be the good general or specific combining ability of PHE67,including its ability to produce hybrids with the approximate maturityand/or hybrid combination traits described herein under the detaileddescription of inbred PHE67. The PHE67 progeny plants that exhibit oneor more of the desired PHE67 traits, such as those listed herein, wouldbe selected and each plant would be harvested separately. This F3 seedfrom each plant would be grown in individual rows and allowed to self.Then selected rows or plants from the rows would be harvestedindividually. The selections would again be based on visual observation,markers and/or measurements for desirable traits of the plants, such asone or more of the desirable PHE67 traits listed herein. The process ofgrowing and selection would be repeated any number of times until aPHE67 progeny inbred plant is obtained. The PHE67 progeny inbred plantwould contain desirable traits derived from inbred plant PHE67, some ofwhich may not have been expressed by the other maize variety to whichinbred line PHE67 was crossed and some of which may have been expressedby both maize varieties but now would be at a level equal to or greaterthan the level expressed in inbred variety PHE67. However, in each casethe resulting progeny line would benefit from the efforts of theinventor(s), and would not have existed but for the inventor(s) work increating PHE67. The PHE67 progeny inbred plants would have, on average,50% of their nuclear genes derived from inbred line PHE67, but manyindividual plants from the population would have a greater percentage oftheir alleles derived from PHE67. This breeding cycle, of crossing andselfing, and optional selection, may be repeated to produce anotherpopulation of PHE67 progeny maize plants with, on average, 25% of theirnuclear genes derived from inbred line PHE67, but, again, manyindividual plants from the population would have a greater percentage oftheir alleles derived from PHE67. This process can be repeated for athird, fourth, fifth, sixth, seventh or more breeding cycles. Anotherembodiment of the invention is a PHE67 progeny plant that has receivedthe desirable PHE67 traits listed herein through the use of PHE67, whichtraits were not exhibited by other plants used in the breeding process.

Therefore, an embodiment of this invention is a PHE67 progeny maizeplant, wherein at least one ancestor of said PHE67 progeny maize plantis the maize plant or plant part of PHE67, and wherein the pedigree ofsaid PHE67 progeny maize plant is within two breeding crosses of PHE67or a plant that has PHE67 as a parent. The progeny plants, parts andplant cells produced from PHE67 may be further characterized as having apercent marker similarity or identity with PHE67 as described herein.

The previous example can be modified in numerous ways, for instanceselection may or may not occur at every selfing generation, selectionmay occur before or after the actual self-pollination process occurs, orindividual selections may be made by harvesting individual ears, plants,rows or plots at any point during the breeding process described. Doublehaploid breeding methods may be used at any step in the process. Insteadof selfing out of the hybrid produced from the inbred, one could firstcross the hybrid to either a parent line or a different inbred, and thenself out of that cross.

The population of plants produced at each and any cycle of breeding isalso an embodiment of the invention, and on average each such populationwould predictably consist of plants containing approximately 50% of itsgenes from inbred line PHE67 in the first breeding cycle, 25% of itsgenes from inbred line PHE67 in the second breeding cycle, 12.5% of itsgenes from inbred line PHE67 in the third breeding cycle, 6.25% in thefourth breeding cycle, 3.125% in the fifth breeding cycle, and so on.However, in each case the use of PHE67 provides a substantial benefit.The linkage groups of PHE67 would be retained in the progeny lines, andsince current estimates of the maize genome size is about 50,000-80,000genes (Xiaowu, Gai et al., Nucleic Acids Research, 2000, Vol. 28, No. 1,94-96), in addition to non-coding DNA that impacts gene expression, itprovides a significant advantage to use PHE67 as starting material toproduce a line that retains desired genetics or traits of PHE67.

Therefore, an embodiment of the invention is a process for making apopulation of PHE67 progeny inbred maize plants comprising obtaining orproducing a first generation progeny maize seed comprising the plant ofPHE67 as a parent, growing said first generation progeny maize seed toproduce first generation maize plants and obtaining self or sibpollinated seed from said first generation maize plants, and growing theself or sib pollinated seed to obtain a population of PHE67 progenyinbred maize plants.

The population of PHE67 progeny inbred maize plants produced by thismethod are also embodiments of the invention, and such population as awhole will retain the expected genetic contribution of PHE67. An inbredline selected from the population of PHE67 progeny inbred maize plantsproduced by this method is an embodiment, and such line may be furthercharacterized by its molecular marker identity or similarity to PHE67.

In this manner, the invention also encompasses a process for making aPHE67 inbred progeny maize plant comprising the steps of obtaining orproducing a first generation progeny maize seed wherein a parent of saidfirst generation progeny maize seed is a PHE67 plant, growing said firstgeneration progeny maize seed to produce a first generation maize plantand obtaining self or sib pollinated seed from said first generationmaize plant, and producing successive filial generations to obtain aPHE67 inbred progeny maize plant. Also an embodiment of this inventionis the first breeding cycle inbred PHE67 maize plant produced by thismethod.

Crosses to Other Species

The utility of inbred maize line PHE67 also extends to crosses withother species. Commonly, suitable species will be of the familyGraminaceae, and especially of the genera Zea, Tripsacum, Coix,Schlerachne, Polytoca, Chionachne, and Trilobachne, of the tribeMaydeae. Potentially suitable for crosses with PHE67 may be the variousvarieties of grain sorghum, Sorghum bicolor (L.) Moench.

INDUSTRIAL APPLICABILITY

Maize is used as human food, livestock feed, and as raw material inindustry. The food uses of maize, in addition to human consumption ofmaize kernels, include both products of dry- and wet-milling industries.The principal products of maize dry milling are grits, meal and flour.The maize wet-milling industry can provide maize starch, maize syrups,and dextrose for food use. Maize oil is recovered from maize germ, whichis a by-product of both dry- and wet-milling industries.

Maize, including both grain and non-grain portions of the plant, is alsoused extensively as livestock feed, primarily for beef cattle, dairycattle, hogs, and poultry.

Industrial uses of maize include production of ethanol, maize starch inthe wet-milling industry and maize flour in the dry-milling industry.The industrial applications of maize starch and flour are based onfunctional properties, such as viscosity, film formation, adhesiveproperties, and ability to suspend particles. The maize starch and flourhave application in the paper and textile industries. Other industrialuses include applications in adhesives, building materials, foundrybinders, laundry starches, explosives, oil-well muds, and other miningapplications.

Plant parts other than the grain of maize are also used in industry: forexample, stalks and husks are made into paper and wallboard and cobs areused for fuel and to make charcoal.

The seed of inbred maize line PHE67, the plant produced from the inbredseed, the hybrid maize plant produced from the crossing of the inbred,hybrid seed, and various parts of the hybrid maize plant and transgenicversions of the foregoing, can be utilized for human food, livestockfeed, and as a raw material in industry.

REFERENCES

-   Aukerman, M. J. et al. (2003) “Regulation of Flowering Time and    Floral Organ Identity by a MicroRNA and Its APETALA2-like Target    Genes” The Plant Cell 15:2730-2741-   Berry et al., Berry, Don et al., “Assessing Probability of Ancestry    Using Simple Sequence Repeat Profiles: Applications to Maize Hybrids    and Inbreds”, Genetics 161:813-824 (2002)-   Berry et al., “Assessing Probability of Ancestry Using Simple    Sequence Repeat Profiles: Applications to Maize Inbred Lines and    Soybean Varieties” Genetics 165:331-342 (2003)-   Boppenmaier, et al., “Comparisons Among Strains of Inbreds for    RFLPs”, Maize Genetics Cooperative Newsletter, 65:1991, p. 90-   Conger, B. V., et al. (1987) “Somatic Embryogenesis From Cultured    Leaf Segments of Zea Mays”, Plant Cell Reports, 6:345-347-   Duncan, D. R., et al. (1985) “The Production of Callus Capable of    Plant Regeneration From Immature Embryos of Numerous Zea Mays    Genotypes”, Planta, 165:322-332-   Edallo, et al. (1981) “Chromosomal Variation and Frequency of    Spontaneous Mutation Associated with in Vitro Culture and Plant    Regeneration in Maize”, Maydica, XXVI:39-56-   Fehr, Walt, Principles of Cultivar Development, pp. 261-286 (1987)    Green, et al. (1975) “Plant Regeneration From Tissue Cultures of    Maize”, Crop Science, Vol. 15, pp. 417-421-   Green, C. E., et al. (1982) “Plant Regeneration in Tissue Cultures    of Maize” Maize for Biological Research, pp. 367-372-   Hallauer, A. R. et al. (1988) “Corn Breeding” Corn and Corn    Improvement, No. 18, pp. 463-481-   Lee, Michael (1994) “Inbred Lines of Maize and Their Molecular    Markers”, The Maize Handbook, Ch. 65:423-432-   Meghji, M. R., et al. (1984) “Inbreeding Depression, Inbred & Hybrid    Grain Yields, and Other Traits of Maize Genotypes Representing Three    Eras”, Crop Science, Vol. 24, pp. 545-549-   Openshaw, S. J., et al. (1994) “Marker-assisted selection in    backcross breeding”. pp. 41-43. In Proceedings of the Symposium    Analysis of Molecular Marker Data. 5-7 Aug. 1994. Corvallis, Oreg.,    American Society for Horticultural Science/Crop Science Society of    America-   Phillips, et al. (1988) “Cell/Tissue Culture and In Vitro    Manipulation”, Corn & Corn Improvement, 3rd Ed., ASA Publication,    No. 18, pp. 345-387-   Poehlman et al (1995) Breeding Field Crop, 4th Ed., Iowa State    University Press, Ames, Iowa., pp. 132-155 and 321-344-   Rao, K. V., et al., (1986) “Somatic Embryogenesis in Glume Callus    Cultures”, Maize Genetics Cooperative Newsletter, No. 60, pp. 64-65-   Sass, John F. (1977) “Morphology”, Corn & Corn Improvement, ASA    Publication, Madison, Wis. pp. 89-109-   Smith, J. S. C., et al., “The Identification of Female Selfs in    Hybrid Maize: A Comparison Using Electrophoresis and Morphology”,    Seed Science and Technology 14, 1-8-   Songstad, D. D. et al. (1988) “Effect of    ACC(1-aminocyclopropane-1-carboyclic acid), Silver Nitrate &    Norbonadiene on Plant Regeneration From Maize Callus Cultures”,    Plant Cell Reports, 7:262-265-   Tomes, et al. (1985) “The Effect of Parental Genotype on Initiation    of Embryogenic Callus From Elite Maize (Zea Mays L.) Germplasm”,    Theor. Appl. Genet., Vol. 70, p. 505-509-   Troyer, et al. (1985) “Selection for Early Flowering in Corn: 10    Late Synthetics”, Crop Science, Vol. 25, pp. 695-697-   Umbeck, et al. (1983) “Reversion of Male-Sterile T-Cytoplasm Maize    to Male Fertility in Tissue Culture”, Crop Science, Vol. 23, pp.    584-588-   Wan et al., “Efficient Production of Doubled Haploid Plants Through    Colchicine Treatment of Anther-Derived Maize Callus”, Theoretical    and Applied Genetics, 77:889-892, 1989-   Wright, Harold (1980) “Commercial Hybrid Seed Production”,    Hybridization of Crop Plants, Ch. 8:161-176-   Wych, Robert D. (1988) “Production of Hybrid Seed”, Corn and Corn    Improvement, Ch. 9, pp. 565-607

DEPOSITS

Applicant has made a deposit of at least 2500 seeds of In bred MaizeLine PHE67 with the American Type Culture Collection (ATCC), Manassas,Va. 20110 USA, ATCC Deposit No. PTA-7758. The seeds deposited with theATCC on Aug. 9, 2006 were taken from the deposit maintained by PioneerHi-Bred International, Inc., 7250 NW 62^(nd) Avenue, Johnston, Iowa,50131 since prior to the filing date of this application. Access to thisdeposit will be available during the pendency of the application to theCommissioner of Patents and Trademarks and persons determined by theCommissioner to be entitled thereto upon request. Upon allowance of anyclaims in the application, the Applicant will make the deposit availableto the public pursuant to 37 C.F.R. §1.808. This deposit of the InbredMaize Line PHE67 will be maintained in the ATCC depository, which is apublic depository, for a period of 30 years, or 5 years after the mostrecent request, or for the enforceable life of the patent, whichever islonger, and will be replaced if it becomes nonviable during that period.Additionally, Applicant has or will satisfy all of the requirements of37 C.F.R. §§1.801 -1.809, including providing an indication of theviability of the sample upon deposit. Applicant has no authority towaive any restrictions imposed by law on the transfer of biologicalmaterial or its transportation in commerce. Applicant does not waive anyinfringement of rights granted under this patent or under the PlantVariety Protection Act (7 USC 2321 et seq.). U.S. Plant VarietyProtection of Inbred Maize Line PHE67 has been applied for. Unauthorizedseed multiplication prohibited.

TABLE 1 VARIETY DESCRIPTION INFORMATION PHE67 AVG STDEV N 1. TYPE:(Describe intermediate types in comments section) 1 = Sweet, 2 = Dent, 3= Flint, 4 = Flour, 5 = Pop and 3 6 = Ornamental. Comments: Flint-Dent2. MATURITY: DAYS HEAT UNITS Days H. Units Emergence to 50% of plants insilk 62 1,367 Emergence to 50% of plants in pollen shed 62 1,354 10% to90% pollen shed 2 42 50% Silk to harvest at 25% moisture 3. PLANT: PlantHeight (to tassel tip) (cm) 239.8 22.64 10 Ear Height (to base of topear node) (cm) 73.6 15.60 10 Length of Top Ear Internode (cm) 16.0 1.3310 Average Number of Tillers per Plant 0.0 0.01 2 Average Number of Earsper Stalk 1.1 0.03 2 Anthocyanin of Brace Roots: 1 = Absent, 2 = Faint,2 3 = Moderate, 4 = Dark 4. LEAF: Width of Ear Node Leaf (cm) 9.0 0.6710 Length of Ear Node Leaf (cm) 72.1 3.78 10 Number of Leaves above TopEar 7.0 0.67 10 Leaf Angle: (at anthesis, 2nd leaf above ear to 23.63.34 10 stalk above leaf) (Degrees) *Leaf Color: V. Dark Green Munsell:7.5GY36 Leaf Sheath Pubescence: 1 = none to 9 = like peach fuzz 3 5.TASSEL: Number of Primary Lateral Branches 2.3 0.48 10 Branch Angle fromCentral Spike 17.2 4.37 10 Tassel Length: (from peduncle node to tasseltip), (cm). 59.8 3.65 10 Pollen Shed: 0 = male sterile, 9 = heavy shed 3*Anther Color: Red Munsell: 7.5RP38 *Glume Color: Dark Green Munsell:7.5GY46 *Bar Glumes (glume bands): 1 = absent, 2 = present 1 PeduncleLength: (from top leaf node to lower florets or 21.0 3.13 10 branches),(cm). 6a. EAR (Unhusked ear) *Silk color: Light Green Munsell: 2.5GY86(3 days after silk emergence) *Fresh husk color: Med. Green Munsell:5GY78 *Dry husk color: Buff Munsell: 2.5Y8.54 (65 days after 50%silking) Ear position at dry husk stage: 1 = upright, 2 = horizontal, 23 = pendant Husk Tightness: (1 = very loose, 9 = very tight) 6 HuskExtension (at harvest): 1 = short(ears exposed), 2 2 = medium (<8 cm), 3= long (8–10 cm), 4 = v. long (>10 cm) 6b. EAR (Husked ear data) EarLength (cm): 16.4 0.97 10 Ear Diameter at mid-point (mm) 42.0 1.41 10Ear Weight (gm): 138.1 12.81 10 Number of Kernel Rows: 13.4 0.97 10Kernel Rows: 1 = indistinct, 2 = distinct 2 Row Alignment: 1 = straight,2 = slightly curved, 3 = spiral 2 Shank Length (cm): 9.9 1.66 10 EarTaper: 1 = slight cylind., 2 = average, 3 = extreme conic. 2 7. KERNEL(Dried): Kernel Length (mm): 11.3 0.67 10 Kernel Width (mm): 8.7 0.67 10Kernel Thickness (mm): 5.3 0.67 10 Round Kernels (shape grade) (%) 60.28.12 2 Aleurone Color Pattern: 1 = homozygous, 2 = segregating 1*Aleurone Color: Yellow Munsell: 10YR814 *Hard Endo. Color: YellowMunsell: 10YR610 Endosperm Type: 3 1 = sweet (su1), 2 = extra sweet(sh2), 3 = normal starch, 4 = high amylose starch, 5 = waxy starch, 6 =high protein, 7 = high lysine, 8 = super sweet (se), 9 = high oil, 10 =other Weight per 100 Kernels (unsized sample) (gm): 34.0 1.41 2 8. COB:*Cob Diameter at mid-point (mm): 22.5 0.85 10 *Cob Color: Pink OrangeMunsell: 10R48 10. DISEASE RESISTANCE: (Rate from 1 = most-susceptableto 9 = most-resistant. Leave blank if not tested, leave race or strainoptions blank if polygenic.) A. LEAF BLIGHTS, WILTS, AND LOCAL INFECTIONDISEASES Anthracnose Leaf Blight (Colletotrichum graminicola) CommonRust (Puccinia sorghi) Common Smut (Ustilago maydis) Eyespot (Kabatiellazeae) Goss's Wilt (Clavibacter michiganense spp. 4 Gray Leaf Spot(Cercospora zeae-maydis) Helminthosporium Leaf Spot (Bipolaris zeicola)Race: 6 Northern Leaf Blight (Exserohilum turcicum) Race: Southern LeafBlight (Bipolaris maydis) Race: Southern Rust (Puccinia polysora)Stewart's Wilt (Erwinia stewartii) Other (Specify):            B.SYSTEMIC DISEASES Corn Lethal Necrosis (MCMV and MDMV) 3 Head Smut(Sphacelotheca reiliana)(% infected) Maize Chlorotic Dwarf Virus (MDV)Maize Chlorotic Mottle Virus (MCMV) Maize Dwarf Mosaic Virus (MDMV)Sorghum Downy Mildew of Corn (Peronosclerospora sorghi) Other (Specify):           C. STALK ROTS 4 Anthracnose Stalk Rot (Colletotrichumgraminicola) Diplodia Stalk Rot (Stenocarpella maydis) Fusarium StalkRot (Fusarium moniliforme) Gibberella Stalk Rot (Gibberella zeae) Other(Specify):            D. EAR AND KERNEL ROTS Aspergillus Ear and KernelRot (Aspergillus flavus) 3 Diplodia Ear Rot (Stenocarpella maydis) 6Fusarium Ear and Kernel Rot (Fusarium moniliforme) Gibberella Ear Rot(Gibberella zeae) Other (Specify):            11. INSECT RESISTANCE:(Rate from 1 = most-suscept. to 9 = most-resist., leave blank if nottested.) Corn Worm (Helicoverpa zea)     Leaf Feeding     Silk Feeding    Ear Damage Corn Leaf Aphid (Rophalosiphum maydis) Corn Sap Beetle(Capophilus dimidiatus) European Corn Borer (Ostrinia nubilalis) 1st.Generation (Typically whorl leaf feeding) 2nd. Generation (Typicallyleaf sheath-collar feeding)     Stalk Tunneling     cm tunneled/plantFall armyworm (Spodoptera fruqiperda)     Leaf Feeding     Silk Feeding    mg larval wt. Maize Weevil (Sitophilus zeamaize) Northern Rootworm(Diabrotica barberi) Southern Rootworm (Diabrotica undecimpunctata)Southwestern Corn Borer (Diatreaea grandiosella)     Leaf Feeding    Stalk Tunneling     cm tunneled/plant Two-spotted Spider Mite(Tetranychus utricae) Western Rootworm (Diabrotica virgifrea virgifrea)Other (Specify):            12. AGRONOMIC TRAITS: 5 Staygreen (at 65days after anthesis; rate from 1-worst to 9-excellent) % Dropped Ears(at 65 days after anthesis) % Pre-anthesis Brittle Snapping 12 %Pre-anthesis Root Lodging % Post-anthesis Root Lodging (at 65 days afteranthesis) % Post-anthesis Stalk Lodging 5,184.0 Kg/ha (Yield at 12–13%grain moisture) *Munsell Glossy Book of color, (A standard colorreference). Kollmorgen Inst. Corp. New Windsor, NY.

TABLE 2 SSR PROFILE DATA FOR PHE67. Marker Base Bin Name Pairs 1.01BNLG1014 124.0 1.01 TUB1 249.2 1.01 UMC2224 364.2 1.02 BNLG1429 205.21.02 BNLG1953 248.1 1.02 UMC2383 206.4 1.03 BNLG1484 136.2 1.03PHI109275 126.1 1.03 UMC1701 117.7 1.04 BNLG2086 218.2 1.05 UMC1626143.8 1.05 UMC1689 149.8 1.05 UMC1906 158.7 1.05 UMC2025 138.1 1.05UMC2232 127.9 1.06 BNLG1057 274.1 1.06 UMC1254 167.0 1.06 UMC1398 164.31.06 UMC1709 353.8 1.06 UMC1919 157.1 1.06 UMC1924 164.2 1.06 UMC2234144.2 1.06 UMC2237 161.8 1.07 BNLG1556 203.9 1.08 UMC2029 156.9 1.08UMC2181 119.1 1.09 BNLG1331 120.9 1.09 UMC2028 116.5 1.09 UMC2047 133.41.1 PHI308707 131.3 1.1 UMC1885 145.9 1.11 PHI064 94.8 1.11 PHI265454233.9 1.11 UMC2241 333.3 1.11 UMC2242 347.9 2.01 PHI96100 294.0 2.01UMC2363 342.6 2.02 BNLG1017 176.0 2.02 BNLG2277 296.4 2.02 UMC1265 302.12.03 BNLG1064 191.0 2.03 UMC2246 145.2 2.04 BNLG1018 134.2 2.04 PRP2131.2 2.05 UMC1459 95.5 2.05 UMC2252 108.5 2.06 BNLG1138 222.0 2.06BNLG1831 194.3 2.06 UMC1004 160.7 2.06 UMC1658 140.4 2.06 UMC2023 143.92.06 UMC2194 316.0 2.07 PHI251315 124.1 2.08 BNLG1141 157.0 2.08BNLG1940 214.4 2.08 PHI435417 218.0 2.08 UMC1230 313.1 2.08 UMC1745216.0 2.08 UMC1947 141.4 2.09 UMC1525 221.7 2.1 PHI101049 234.1 2.1UMC2184 127.3 3 PHI453121 222.9 3.01 PHI404206 302.8 3.01 UMC1892 105.63.01 UMC2376 161.0 3.02 BNLG1647 137.7 3.02 PHI243966 208.1 3.02 UMC1814339.1 3.02 UMC1886 155.3 3.02 UMC2258 141.2 3.04 BNLG1019A 155.2 3.04TPI4 146.2 3.04 UMC1025 163.6 3.04 UMC1683 86.9 3.04 UMC1908 135.9 3.04UMC2263 393.5 3.05 BNLG1035 112.8 3.05 GST4 185.6 3.05 UMC1307 135.83.05 UMC2265 217.7 3.06 BNLG1160 220.4 3.06 BNLG1951 121.3 3.06 BNLG2241172.7 3.06 PHI102228 127.7 3.06 UMC1876 165.7 3.07 UMC1825 159.9 3.07UMC2050 113.9 3.08 UMC1844 142.9 3.08 UMC1915 95.0 3.08 UMC2276 138.13.09 UMC1813 162.2 4 MTL1 141.1 4.01 UMC1757 197.4 4.03 ADH2 117.7 4.04ZP1 235.2 4.05 BNLG1265 223.9 4.05 BNLG1755 240.3 4.05 GPC1 177.8 4.05UMC1175 281.3 4.05 UMC1662 112.1 4.05 UMC2061 137.1 4.06 UMC1869 151.44.07 BNLG1189 138.2 4.07 UMC1194 167.3 4.07 UMC1651 93.8 4.08 BNLG2162183.3 4.08 BNLG2244 199.6 4.08 SSU1 287.5 4.08 UMC1559 144.3 4.08UMC1667 147.1 4.08 UMC2187 91.1 4.09 UMC1820 142.2 4.09 UMC1940 134.64.09 UMC2046 115.9 4.1 BNLG589 154.5 5 BNLG1006 241.2 5 UMC1240 213.55.03 UMC1686 96.8 5.03 UMC1784 336.9 5.03 UMC2035 102.3 5.03 UMC2295115.2 5.04 UMC1092 134.6 5.04 UMC2302 345.1 5.05 PHI333597 216.4 5.05UMC1687 160.5 5.05 UMC1853 113.7 5.05 UMC2026 100.7 5.06 GLN4 250.1 5.06UMC1941 113.2 5.06 UMC2305 156.5 5.07 BNLG1118 82.7 5.07 UMC2013 125.35.09 UMC2308 150.1 6 GPC2 348.1 6 UMC1883 86.2 6.01 UMC2056 167.8 6.02SAUR1 113.4 6.03 UMC1887 99.3 6.04 UMC1857 150.8 6.04 UMC1918 167.3 6.04UMC2006 174.6 6.04 UMC2317 129.7 6.05 BNLG1174 219.0 6.05 PMG1 147.56.05 UMC1314 346.6 6.05 UMC1805 100.1 6.06 UMC1859 92.8 6.07 BNLG1759A126.1 6.07 PHI299852 117.3 6.07 TLK1 83.1 6.07 UMC1350 120.0 6.07UMC1897 157.0 6.08 UMC2059 127.3 7 UMC1378 118.2 7.01 UMC1632 142.9 7.02CYP6 119.7 7.02 UMC1929 157.2 7.02 UMC2327 136.5 7.03 BNLG1070 145.67.03 UMC1713 139.5 7.03 UMC1841 109.2 7.03 UMC1865 151.6 7.03 UMC1888149.1 7.04 PHI328175 98.3 7.04 UMC1708 332.7 7.05 PHI069 195.2 7.06PHI116 165.0 8.02 BNLG1194 175.4 8.02 UMC1304 246.4 8.03 BNLG2082 173.08.03 PHI100175 145.3 8.03 PHI121 93.6 8.03 UMC1735 104.1 8.04 BNLG2046326.4 8.04 UMC1343 317.3 8.04 UMC1858 128.0 8.05 BNLG1176 218.2 8.05UMC1287 312.7 8.05 UMC1340 212.2 8.05 UMC1864 164.0 8.05 UMC1889 117.58.06 BNLG1152 148.6 8.07 BNLG1828 185.8 8.08 GST1 79.3 8.08 UMC2052146.4 8.09 PHI233376 151.9 8.09 UMC1638 141.1 8.09 UMC1663 208.7 9.01SH1 249.0 9.01 UMC1040 237.1 9.02 UMC1636 188.6 9.02 UMC1698 116.4 9.02UMC1764 306.3 9.03 UMC1420 316.9 9.03 UMC1634 114.0 9.04 BNLG1012 161.69.04 BNLG1159B 148.1 9.04 UMC2398 126.1 9.05 UMC1657 164.6 9.05 UMC2338105.0 9.05 UMC2341 130.1 9.05 UMC2342 135.3 9.06 UMC2346 288.3 9.06UMC2358 129.4 9.07 BNLG1375 141.5 9.07 UMC1675 165.3 9.07 UMC1714 158.89.07 UMC1942 131.7 9.08 BNLG1129 300.7 10.01 UMC2018 162.8 10.01 UMC2053103.7 10.02 PHI059 153.0 10.02 UMC2069 375.2 10.03 BNLG1079 172.5 10.03BNLG1655 126.6 10.03 UMC1345 166.6 10.03 UMC1367 328.0 10.03 UMC1381210.3 10.03 UMC1962 132.5 10.04 MGS1 158.0 10.04 UMC1330 340.6 10.04UMC1827 126.0 10.04 UMC1911 159.9 10.04 UMC1930 125.2 10.05 BNLG1074178.0 10.06 BNLG1028 140.0 10.06 UMC1993 127.3 10.07 BNLG1450 178.210.07 UMC1556 254.4 10.07 UMC1640 103.8 10.07 UMC1645 164.0

TABLE 3A PAIRED INBRED COMPARISON REPORT Variety #1: PHE67 Variety #2:PH4GP YIELD YIELD TSTWT EGRWTH ESTCNT BU/A 56# BU/A 56# MST PCT LB/BUSCORE COUNT Stat ABS % MN ABS ABS ABS ABS Mean1 87.6 88.3 19.6 59.0 6.530.2 Mean2 83.5 84.4 20.6 58.0 5.5 25.8 Locs 11 11 12 2 10 23 Reps 20 2021 4 10 24 Diff 4.1 4.0 1.0 1.1 1.0 4.4 Prob 0.544 0.563 0.009 0.1030.004 0.001 TILLER GDUSHD GDUSLK POLWT POLWT TASSZ PCT GDU GDU VALUEVALUE SCORE Stat ABS ABS ABS ABS % MN ABS Mean1 1.0 137.1 139.4 63.651.6 2.9 Mean2 3.8 139.8 139.4 56.2 45.8 2.0 Locs 22 63 62 6 6 52 Reps22 63 62 10 10 52 Diff 2.8 −2.7 0.0 7.4 5.8 0.9 Prob 0.013 0.000 0.9770.458 0.403 0.000 PLTHT EARHT STAGRN SCTGRN TEXEAR BARPLT CM CM SCORESCORE SCORE % NOT Stat ABS ABS ABS ABS ABS ABS Mean1 237.7 80.1 4.8 7.27.0 95.5 Mean2 223.6 83.2 3.9 6.4 7.0 96.0 Locs 40 19 10 5 1 30 Reps 4019 12 5 1 30 Diff 14.1 −3.1 0.9 0.8 0.0 −0.4 Prob 0.000 0.374 0.0540.374 — 0.778 GLFSPT NLFBLT SLFBLT GOSWLT STWWLT ANTROT SCORE SCORESCORE SCORE SCORE SCORE Stat ABS ABS ABS ABS ABS ABS Mean1 3.6 6.9 5.05.5 5.0 4.1 Mean2 3.2 5.6 6.0 7.5 4.5 3.9 Locs 13 4 1 1 2 5 Reps 21 8 22 2 10 Diff 0.5 1.3 −1.0 −2.0 0.5 0.2 Prob 0.111 0.015 — — 0.500 0.704FUSERS GIBERS DIPERS COMRST CLDTST CLDTST SCORE SCORE SCORE SCORE PCTPCT Stat ABS ABS ABS ABS ABS % MN Mean1 6.5 5.0 3.4 6.7 90.7 96.6 Mean25.9 6.5 3.9 6.0 84.9 90.1 Locs 7 1 6 3 7 7 Reps 12 2 12 3 7 7 Diff 0.6−1.5 −0.5 0.7 5.9 6.5 Prob 0.049 — 0.524 0.184 0.068 0.068 KSZDCD HDSMTERTLPN LRTLPN STLLPN PCT % NOT % NOT % NOT % NOT Stat ABS ABS ABS ABSABS Mean1 3.3 96.9 79.0 100.0 30.0 Mean2 2.6 87.8 84.0 100.0 35.0 Locs 85 4 1 1 Reps 8 10 5 2 2 Diff 0.7 9.2 −5.0 0.0 −5.0 Prob 0.488 0.1030.604 — —

TABLE 3B PAIRED INBRED COMPARISON REPORT Variety #1: PHE67 Variety #2:PHCER YIELD YIELD TSTWT EGRWTH ESTCNT BU/A 56# BU/A 56# MST PCT LB/BUSCORE COUNT Stat ABS % MN ABS ABS ABS ABS Mean1 79.8 82.6 18.7 59.1 6.433.2 Mean2 101.9 106.5 18.9 59.0 6.4 34.8 Locs 24 24 28 4 16 28 Reps 4848 52 8 17 41 Diff −22.1 −23.9 0.2 0.1 0.0 −1.6 Prob 0.000 0.000 0.5580.866 0.868 0.059 TILLER GDUSHD GDUSLK POLWT POLWT TASSZ PCT GDU GDUVALUE VALUE SCORE Stat ABS ABS ABS ABS % MN ABS Mean1 1.1 137.0 139.354.5 44.2 3.0 Mean2 1.5 136.9 137.4 54.1 43.0 2.8 Locs 28 88 86 7 7 74Reps 28 88 86 13 13 74 Diff 0.4 0.1 2.0 0.4 1.2 0.2 Prob 0.557 0.7690.000 0.937 0.796 0.068 PLTHT EARHT STAGRN BRTSTK SCTGRN TEXEAR CM CMSCORE % NOT SCORE SCORE Stat ABS ABS ABS ABS ABS ABS Mean1 236.6 83.14.8 99.0 7.3 4.5 Mean2 230.1 88.8 4.4 98.1 8.0 3.0 Locs 59 34 13 1 4 2Reps 59 34 15 2 4 2 Diff 6.5 −5.7 0.5 1.0 −0.8 1.5 Prob 0.002 0.0060.111 — 0.058 0.205 BARPLT GLFSPT NLFBLT SLFBLT GOSWLT STWWLT % NOTSCORE SCORE SCORE SCORE SCORE Stat ABS ABS ABS ABS ABS ABS Mean1 95.83.6 6.4 5.3 5.5 5.3 Mean2 98.2 3.8 7.0 4.5 7.0 5.3 Locs 43 16 6 2 1 3Reps 44 24 12 4 2 3 Diff −2.4 −0.2 −0.6 0.8 −1.5 0.0 Prob 0.015 0.4810.135 0.500 — 1.000 ANTROT FUSERS GIBERS DIPERS COMRST CLDTST SCORESCORE SCORE SCORE SCORE PCT Stat ABS ABS ABS ABS ABS ABS Mean1 3.9 6.35.0 3.4 6.7 93.2 Mean2 2.9 6.8 8.5 5.3 7.3 91.7 Locs 5 13 1 6 3 15 Reps10 19 2 12 3 15 Diff 1.0 −0.5 −3.5 −1.8 −0.7 1.5 Prob 0.022 0.139 —0.110 0.184 0.083 CLDTST KSZDCD HDSMT ERTLPN LRTLPN STLLPN PCT PCT % NOT% NOT % NOT % NOT Stat % MN ABS ABS ABS ABS ABS Mean1 99.1 3.1 96.9 86.0100.0 30.0 Mean2 97.5 4.1 95.6 96.0 67.5 37.5 Locs 15 16 5 6 2 1 Reps 1516 10 8 4 2 Diff 1.6 −1.0 1.3 −10.0 32.5 −7.5 Prob 0.087 0.118 0.6910.511 0.386 —

TABLE 4 GENERAL COMBINING ABILITY REPORT FOR PHE67 PRM Day ABS Mean 104PRM Day ABS Reps 886 PRMSHD Day ABS Mean 104 PRMSHD Day ABS Reps 796YIELD bu/a 56# ABS Mean 193.3 YIELD bu/a 56# ABS Reps 483 YIELD bu/a 56#ABS Years 3 YIELD bu/a 56# % MN Mean 103.5 YIELD bu/a 56# % MN Reps 483MST pct ABS Mean 20.8 MST pct ABS Reps 487 MST pct % MN Mean 101.2 MSTpct % MN Reps 487 YLDMST ABS Mean 102.2 YLDMST ABS Reps 489 STLPCN % NOT% MN Mean 110 STLPCN % NOT % MN Reps 58 STLLPN % NOT % MN Mean 112STLLPN % NOT % MN Reps 83 ERTLPN % NOT % MN Mean 109 ERTLPN % NOT % MNReps 10 LRTLPN % NOT % MN Mean 105 LRTLPN % NOT % MN Reps 49 TSTWT lb/bu% MN Mean 100 TSTWT lb/bu % MN Reps 383 STKCNT count % MN Mean 100STKCNT count % MN Reps 859 PLTHT in % MN Mean 101 PLTHT in % MN Reps 149EARHT in % MN Mean 100 EARHT in % MN Reps 143 BRTSTK % NOT % MN Mean 101BRTSTK % NOT % MN Reps 17 BORBMN % NOT % MN Mean 68 BORBMN % NOT % MNReps 26 BRENGMN kg * mm Mean 103 % MN BRENGMN kg * mm Reps 3 % MN GLFSPTscore ABS Mean 5 GLFSPT score ABS Reps 41 STAGRN score ABS Mean 6 STAGRNscore ABS Reps 100 HSKCVR score ABS Mean 5 HSKCVR score ABS Reps 17ECBLSI score ABS Mean 5.7 ECBLSI score ABS Reps 16

TABLE 5A INBREDS IN HYBRID COMBINATION REPORT Variety #1: HYBRIDCONTAINING PHE67 Variety #2: 35A30 YIELD YIELD MST ESTCNT GDUSHD GDUSLKBU/A 56# BU/A 56# PCT COUNT GDU GDU Stat ABS % MN % MN % MN % MN % MNMean1 200.0 105.4 103.1 97.7 101.0 101.5 Mean2 207.4 109.3 101.3 99.3102.7 102.3 Locs 46 46 46 4 14 14 Reps 111 111 111 12 26 26 Diff −7.4−3.8 −1.8 −1.6 −1.7 −0.8 Prob 0.009 0.016 0.031 0.728 0.005 0.095 STKCNTPLTHT EARHT STAGRN STKLDG STLLPN COUNT CM CM SCORE % NOT % NOT Stat % MN% MN % MN % MN % MN % MN Mean1 99.6 102.1 99.2 132.9 137.8 131.2 Mean2100.7 108.0 102.9 108.6 85.0 114.3 Locs 82 13 13 11 1 5 Reps 188 31 3124 2 14 Diff −1.1 −5.9 −3.7 24.3 52.8 16.9 Prob 0.033 0.000 0.094 0.008— 0.230 DRPEAR TSTWT GLFSPT NLFBLT GOSWLT ANTROT % NOT LB/BU SCORE SCORESCORE SCORE Stat % MN ABS ABS ABS ABS ABS Mean1 100.2 56.2 4.5 6.0 7.37.0 Mean2 99.7 56.8 3.9 6.3 7.0 3.0 Locs 1 43 4 2 2 1 Reps 3 105 8 4 4 2Diff 0.5 −0.6 0.6 −0.3 0.3 4.0 Prob — 0.000 0.080 0.500 0.795 — FUSERSGIBERS DIPERS ECB1LF ECB2SC HSKCVR SCORE SCORE SCORE SCORE SCORE SCOREStat ABS ABS ABS ABS ABS ABS Mean1 4.0 8.0 3.3 8.8 8.7 4.2 Mean2 5.0 6.54.3 5.3 5.2 4.7 Locs 2 1 2 2 3 3 Reps 4 2 4 4 6 6 Diff −1.0 1.5 −1.0 3.53.5 −0.6 Prob 0.500 — 1.000 0.177 0.026 0.560 GIBROT BRTSTK HDSMT LRTLPNSTLPCN SCORE % NOT % NOT % NOT % NOT Stat ABS ABS ABS ABS ABS Mean1 7.095.3 97.4 99.4 97.5 Mean2 3.5 93.6 97.4 94.8 87.9 Locs 1 2 2 4 4 Reps 24 6 9 11 Diff 3.5 1.7 −0.1 4.6 9.6 Prob — 0.751 0.858 0.115 0.059

TABLE 5B INBREDS IN HYBRID COMBINATION REPORT Variety #1: HYBRIDCONTAINING PHE67 Variety #2: 36W66 YIELD YIELD MST ESTCNT GDUSHD GDUSLKBU/A 56# BU/A 56# PCT COUNT GDU GDU Stat ABS % MN % MN % MN % MN % MNMean1 199.6 105.5 103.1 97.7 101.0 101.5 Mean2 187.6 99.2 98.8 99.5 99.2100.4 Locs 47 47 47 4 14 14 Reps 112 112 112 11 26 26 Diff 12.1 6.3 −4.3−1.9 1.8 1.1 Prob 0.000 0.000 0.000 0.634 0.000 0.018 STKCNT PLTHT EARHTSTAGRN STKLDG STLLPN COUNT CM CM SCORE % NOT % NOT Stat % MN % MN % MN %MN % MN % MN Mean1 99.6 102.1 99.2 132.9 137.8 131.2 Mean2 100.7 98.498.5 119.3 124.0 119.0 Locs 82 13 13 11 1 5 Reps 188 31 31 24 2 14 Diff−1.1 3.7 0.7 13.6 13.8 12.2 Prob 0.069 0.003 0.742 0.014 — 0.138 DRPEARTSTWT GLFSPT NLFBLT GOSWLT ANTROT % NOT LB/BU SCORE SCORE SCORE SCOREStat % MN ABS ABS ABS ABS ABS Mean1 100.2 56.2 4.5 6.0 7.3 7.0 Mean2100.2 55.8 4.0 5.3 7.5 4.0 Locs 1 44 4 2 2 1 Reps 3 106 8 4 4 1 Diff 0.00.5 0.5 0.8 −0.3 3.0 Prob — 0.001 0.182 0.205 0.874 — FUSERS GIBERSDIPERS ECB1LF ECB2SC HSKCVR SCORE SCORE SCORE SCORE SCORE SCORE Stat ABSABS ABS ABS ABS ABS Mean1 4.0 8.0 3.3 8.8 8.7 4.2 Mean2 5.0 5.0 2.8 6.34.3 5.4 Locs 2 1 2 2 3 3 Reps 4 2 4 4 6 6 Diff −1.0 3.0 0.5 2.5 4.3 −1.3Prob 1.000 — 0.500 0.242 0.069 0.145 GIBROT BRTSTK HDSMT LRTLPN STLPCNSCORE % NOT % NOT % NOT % NOT Stat ABS ABS ABS ABS ABS Mean1 7.0 95.397.4 99.4 97.5 Mean2 6.5 98.8 94.9 96.0 90.8 Locs 1 2 2 4 4 Reps 2 4 6 912 Diff 0.5 −3.5 2.5 3.4 6.7 Prob — 0.317 0.600 0.286 0.139

TABLE 6 PHENOTYPIC DATA FROM HYBRIDS PRODUCED WITH PHE67. PRM PRM YIELDYIELD YIELD YIELD YIELD PRM PRM SHD SHD bu/a bu/a bu/a bu/a bu/a Day DayDay Day 56# 56# 56# 56# 56# ABS ABS ABS ABS ABS ABS ABS % MN % MN MEANREPS MEAN REPS MEAN REPS YRS MEAN REPS Hybrid 1 103 619 104 607 190.9335 3 102.7 335 Hybrid 2 104 189 103 189 197.9 104 1 105 104 Hybrid 3112  78 200  44 2 106.8  44 YLD STL STL MST MST MST MST MST YLD MST PCNPCN pct pct pct pct Value Value % NOT % NOT ABS ABS % MN % MN ABS ABS %MN % MN MEAN REPS MEAN REPS MEAN REPS MEAN REPS Hybrid 1 20.9 339 100.5339 102 341 108 37 Hybrid 2 20.3 104 103.1 104 101.8 104 111 12 Hybrid 321.1 44 101.9 44 104.8 44 118 9 STL STL ERT ERT LRT LRT TST TST LPN LPNLPN LPN LPN LPN WT WT % NOT % NOT % NOT % NOT % NOT % NOT lb/bu lb/bu %MN % MN % MN % MN % MN % MN % MN % MN MEAN REPS MEAN REPS MEAN REPS MEANREPS Hybrid 1 108 57 110 6 105 32 100.4 248 Hybrid 2 131 13 109 8 99.3102 Hybrid 3 111 13 109 4 102 9 99.1 33 STK STK BRT BRT CNT CNT PLTHTPLTHT STK STK count count in in EAR HT EAR HT % NOT % NOT % MN % MN % MN% MN in % MN in % MN % MN % MN MEAN REPS MEAN REPS MEAN REPS MEAN REPSHybrid 1 100 622 100 107 99 101 101 13 Hybrid 2 100 166 102 25 100 25 984 Hybrid 3 101 71 103 17 103 17 BOR BOR BREN BREN GLF GLF BMN BMN GMNGMN SPT SPT % NOT % NOT kg *mm kg *mm score score % MN % MN % MN % MNABS ABS MEAN REPS MEAN REPS MEAN REPS Hybrid 1 66  20 103.1 3 5 28 Hybrid 2 5 8 Hybrid 3 78   6 5 5 STA STA ECB ECB GRN GRN HSK CVR HSK CVRLSI LSI score score score score score score ABS ABS ABS ABS ABS ABS MEANREPS MEAN REPS MEAN REPS Hybrid 1 6 64 5 11  5.6 11  Hybrid 2 7 21 3 46.7 3 Hybrid 3 6 15 7 2 4.5 2

All publications, patents and patent applications mentioned in thespecification are indicative of the level of those skilled in the art towhich this invention pertains. All such publications, patents and patentapplications are incorporated by reference herein for the purpose citedto the same extent as if each was specifically and individuallyindicated to be incorporated by reference herein.

The foregoing invention has been described in detail by way ofillustration and example for purposes of clarity and understanding. Asis readily apparent to one skilled in the art, the foregoing are onlysome of the methods and compositions that illustrate the embodiments ofthe foregoing invention. It will be apparent to those of ordinary skillin the art that variations, changes, modifications and alterations maybe applied to the compositions and/or methods described herein withoutdeparting from the true spirit, concept and scope of the invention.

1. A seed of maize inbred line designated PHE67, representative seed ofsaid line having been deposited under ATCC Accession number PTA-7758. 2.A maize plant, or a part thereof, produced by growing the seed ofclaim
 1. 3. Pollen of the plant of claim
 2. 4. An ovule or ovules of theplant of claim
 2. 5. The maize plant of claim 2, wherein said plant hasbeen detasseled.
 6. A maize plant, or a part thereof, expressing all thephysiological and morphological characteristics of inbred line PHE67,representative seed of said line having been deposited under ATCCAccession number PTA-7758.
 7. A maize plant cell comprising all of thealleles of inbred line PHE67 at the SSR loci listed in Table 2,representative seed of said line having been deposited under ATCCAccession number PTA-7758.
 8. A maize seed comprising the maize plantcell of claim
 7. 9. A maize plant, or a part thereof, produced bygrowing the seed of claim
 8. 10. A tissue culture comprising the maizeplant cell of claim
 7. 11. A tissue culture of regenerable cellsproduced from the plant of claim
 2. 12. Protoplasts or callus producedfrom the tissue culture of claim
 11. 13. The tissue culture of claim 11,wherein the regenerable cells of the tissue culture are produced fromprotoplasts or from tissue of a plant part selected from the groupconsisting of leaf, pollen, embryo, immature embryo, meristematic cells,immature tassels, microspores, root, root tip, anther, silk, flower,kernel, ear, cob, husk and stalk.
 14. A maize plant regenerated from thetissue culture of claim 13, said plant having all the morphological andphysiological characteristics of maize inbred line PHE67, representativeseed of said line having been deposited under ATCC Accession numberPTA-7758.
 15. A process for producing an F1 hybrid maize seed, saidprocess comprising crossing the plant of claim 2 with a different maizeplant and harvesting F1 hybrid maize seed.
 16. The process of claim 15,further comprising growing the F1 hybrid maize seed to produce a hybridmaize plant.
 17. The maize plant according to claim 2, wherein saidplant is modified by the addition of at least one mutant or transgenicgene that confers a characteristic selected from the group consisting ofmale sterility, site-specific recombination, and abiotic stresstolerance.
 18. The maize plant of claim 17, wherein said characteristicis male sterility and the male sterility is conferred by a nucleic acidmolecule that confers male sterility.
 19. The maize plant of claim 17,wherein said characteristic is site-specific recombination and thesite-specific recombination is conferred by a member of the groupconsisting of flp/frt, cre/lox, Gin, Pin, and R/RS.
 20. The maize plantof claim 17, wherein said characteristic is abiotic stress tolerance andthe abiotic stress tolerance is conferred by a nucleic acid that conferssaid abiotic stress tolerance.
 21. The maize plant according to claim 2,wherein said plant is modified by the addition of at least one mutant ortransgenic gene that confers a characteristic selected from the groupconsisting of resistance comprises herbicide resistance, diseaseresistance and insect resistance.
 22. The maize plant of claim 21,wherein said characteristic is herbicide resistance and the herbicide isglyphosate, glufosinate, a sulfonylurea herbicide, an imidazol inoneherbicide, a hydroxyphenylpyruvate dioxygenase inhibitor or aprotoporphyrinogen oxidase inhibitor.
 23. The maize plant of claim 21,wherein said characteristic is insect resistance and the insectresistance is conferred by a nucleic acid molecule encoding a Bacillusthuringiensis endotoxin.
 24. The maize plant of claim 21, wherein saidcharacteristic is disease resistance and the disease is caused by abacterium, fungus, nematode or virus.
 25. The maize plant according toclaim 2, wherein said plant is modified by the addition of at least onemutant or transgenic gene that confers a characteristic selected fromthe group consisting of altered phosphorus, altered antioxidants,altered fatty acids, altered essential amino acids and alteredcarbohydrates.
 26. The maize plant of claim 25, wherein saidcharacteristic is conferred by a nucleic acid molecule selected from thegroup consisting of NTR, TRX, dek1, TUSC 27, cs27, en27, Ipa1, Ipa3,mi1ps, hpt and hggt.
 27. A process of introducing a desired trait intomaize inbred line PHE67 comprising: (a) crossing PHE67 plants grown fromPHE67 seed, representative seed of which has been deposited under ATCCAccession number PTA-7758, with plants of another maize line thatcomprise a desired trait to produce F1 progeny plants, wherein thedesired trait is selected from the group consisting of male sterility,site-specific recombination, increased transformability and abioticstress tolerance; (b) selecting F1 progeny plants that have the desiredtrait to produce selected F1 progeny plants; (c) crossing the selectedprogeny plants with the PHE67 plants to produce backcross progenyplants; (d) selecting for backcross progeny plants that have the desiredtrait and physiological and morphological characteristics of maizeinbred line PHE67 to produce selected backcross progeny plants; and (e)repeating steps (c) and (d) three or more times in succession to produceselected fourth or higher backcross progeny plants that comprise thedesired trait and all of the physiological and morphologicalcharacteristics of maize inbred line PHE67 listed in Table 1 asdetermined at the 5% significance level when grown in the sameenvironmental conditions.
 28. A maize plant produced by the process ofclaim 27, wherein the plant has the desired trait and all of thephysiological and morphological characteristics of maize inbred linePHE67 listed in Table 1 as determined at the 5% significance level whengrown in the same environmental conditions.
 29. The maize plant of claim28, wherein the desired trait is male sterility and the trait isconferred by a nucleic acid molecule that confers male sterility. 30.The maize plant of claim 28, wherein the desired trait is site specificrecombination and the site-specific recombination is conferred by amember of the group consisting of flp/frt, cre/lox, Gin, Pin, and R/RS.31. The maize plant of claim 28, wherein the desired trait is abioticstress tolerance and the trait is conferred by a nucleic acid thatconfers said abiotic stress tolerance.
 32. The maize plant of claim 28,wherein the desired trait is increased transformability and the trait isconferred by inbred maize line Hi-II.
 33. A process of introducing adesired trait into maize inbred line PHE67 comprising: (a) crossingPHE67 plants grown from PHE67 seed, representative seed of which hasbeen deposited under ATCC Accession number PTA-7758, with plants ofanother maize line that comprise a desired trait to produce F1 progenyplants, wherein the desired trait is selected from the group consistingof herbicide resistance, insect resistance and disease resistance; (b)selecting F1 progeny plants that have the desired trait to produceselected F1 progeny plants; (c) crossing the selected progeny plantswith the PHE67 plants to produce backcross progeny plants; (d) selectingfor backcross progeny plants that have the desired trait andphysiological and morphological characteristics of maize inbred linePHE67 to produce selected backcross progeny plants; and (e) repeatingsteps (c) and (d) three or more times in succession to produce selectedfourth or higher backcross progeny plants that comprise the desiredtrait and all of the physiological and morphological characteristics ofmaize inbred line PHE67 listed in Table 1 as determined at the 5%significance level when grown in the same environmental conditions. 34.A maize plant produced by the process of claim 33, wherein the plant hasthe desired trait and all of the physiological and morphologicalcharacteristics of maize inbred line PHE67 listed in Table 1 asdetermined at the 5% significance level when grown in the sameenvironmental conditions.
 35. The maize plant of claim 34, wherein thedesired trait is herbicide resistance and said herbicide is glyphosate,glufosinate, a sulfonylurea herbicide, an imidazolinone herbicide, ahydroxyphenylpyruvate dioxygenase inhibitor or a protoporphyrinogenoxidase inhibitor.
 36. The maize plant of claim 34, wherein the desiredtrait is insect resistance and the insect resistance is conferred by anucleic acid molecule encoding a Bacillus thuringiensis endotoxin. 37.The maize plant of claim 34, wherein the desired trait is diseaseresistance and the disease is caused by a bacterium, fungus, nematode orvirus.
 38. A process of introducing altered grain characteristics traitinto maize inbred line PHE67 comprising: (a) crossing PHE67 plants grownfrom PHE67 seed, representative seed of which has been deposited underATCC Accession number PTA-7758, with plants of another maize line thatcomprise a desired trait to produce F1 progeny plants, wherein thedesired trait is altered phosphorus, altered antioxidants, altered fattyacids, altered essential amino acids or altered carbohydrates; (b)selecting F1 progeny plants that have the desired trait to produceselected F1 progeny plants; (c) crossing the selected progeny plantswith the PHE67 plants to produce backcross progeny plants; (d) selectingfor backcross progeny plants that have the desired trait andphysiological and morphological characteristics of maize inbred linePHE67 to produce selected backcross progeny plants; and (e) repeatingsteps (c) and (d) three or more times in succession to produce selectedfourth or higher backcross progeny plants that comprise the desiredtrait and all of the physiological and morphological characteristics ofmaize inbred line PHE67 listed in Table 1 as determined at the 5%significance level when grown in the same environmental conditions. 39.A maize plant produced by the process of claim 38, wherein the plant hasthe desired trait and all of the physiological and morphologicalcharacteristics of maize inbred line PHE67 listed in Table 1 asdetermined at the 5% significance level when grown in the sameenvironmental conditions.
 40. The maize plant of claim 39, wherein thedesired trait is altered carbohydrate and the carbohydrate is waxystarch.